Fluorescent quenching detection reagents and methods

ABSTRACT

Oligonucleotide-fluorophore-quencher conjugates wherein the fluorophore moiety has emission wavelengths in the range of about (300) to about (800) nm, and or where the quencher includes a substituted 4-(phenyldiazenyl)phenylamine structure provide improved signal to noise ratios and other advantageous characteristics in hybridization and related assays. The oligonucleotide-fluorophore-quencher conjugates can be synthesized by utilizing novel phosphoramidite reagents that incorporate the quencher moiety based on the substituted 4-(phenyldiazenyl)phenylamine structure, and or novel phosphoramidite reagents that incorporate a fluorophore moiety based on the substituted coumarin, substituted 7-hydroxy-3H-phenoxazin-3-one, or substituted 5,10-dihydro-10 [phenyl]pyrido[2,3-d;6,5-d′]dipyrimidine-2,4,6,8-(1H, 3H, 7H, 9H, 10H)-tetrone structure. Oligonucleotide-fluorophore-quencher-minor groove binder conjugates including a pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indolin-7-caroxylate (DPI 3 ) moiety as the minor groove binder and the substituted 4-(phenyldiazenyl)phenylamine moiety as the quencher, were synthesized and have substantially improved hybridization and signal to noise ratio properties.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to oligonucleotide-quencher-fluorescent-dyeconjugates having improved characteristics, and to reagents suitable forincorporating novel quencher and fluorescent dye moieties intooligonucleotides. The invention also relates to the use ofoligonucleotide- quencher-fluorescent-dye conjugates in detectionmethods for nucleic acid targets.

[0003] 2. Brief Description of Related Art

[0004] Synthetic oligonucleotides have been used for years as sequencespecific probes for complementary DNA and RNA targets. These methodshave broad application in forensics, molecular biology and medicaldiagnostics since they allow the identification and quantitation ofspecific nucleic acid targets. Early uses of DNA probes relied onradioactivity (typically ³²p) as the label, while recent methods usereporter molecules which include chemiluminescent and fluorescentgroups. Improved instrumentation has allowed the sensitivity of thesespectroscopic methods to approach or surpass the radiolabeled methods.Recently developed detection methods employ the process of fluorescenceresonance energy transfer (FRET) for the detection of probehybridization rather than direct detection of fluorescence intensity. Inthis type of assay, FRET occurs between a donor fluorophore (reporter)and an acceptor molecule (quencher) when the absorption spectrum of thequencher molecule overlaps with the emission spectrum of the donorfluorophore and the two molecules are in close proximity. Theexcited-state energy of the donor fluorophore is transferred to theneighboring acceptor by a resonance dipole-induced dipole interaction,which results in quenching of the donor fluorescence. If the acceptormolecule is a fluorophore, its fluorescence may sometimes be increased.The efficiency of the energy transfer between the donor and acceptormolecules is highly dependent on distance between the molecules.Equations describing this relationship are known. The Forster distance(R_(o)) is described as the distance between the donor and acceptormolecules where the energy transfer is 50% efficient. Other mechanismsof fluorescence quenching are also known, such as, collisional andcharge transfer quenching.

[0005] Typically detection methods based on FRET are designed in such away that the donor fluorophore and acceptor molecules are in closeproximity so that quenching of the donor fluorescence is efficient.During the assay, the donor and acceptor molecules are separated suchthat fluorescence occurs. FRET-based detection assays have beendeveloped in the fields of nucleic acid hybridization and enzymology.Several forms of the FRET hybridization assays are reviewed (NonisotopicDNA Probe Techniques, Academic Press, Inc., San Diego 1992, pp.311-352).

[0006] Since its discovery, the polymerase chain reaction (PCR) hasrevolutionized molecular biology. This technique allows amplification ofspecific DNA sequences, thus allowing DNA probe assays to be executedfrom a single DNA target copy. PCR-based diagnostic assays haveinitially not been used routinely, in part due to problems with samplehandling and possible contamination with non-source DNA. Recently, newhomogeneous fluorescent-based DNA assays have been described which candetect the progress of PCR as it occurs (“real-time” PCR detection)using spectrofluorometric temperature cyclers. Two popular assay formatsuse DNA probes which become fluorescent as DNA amplification occurs(fluorogenic probes).

[0007] The first format for “real-time” PCR uses DNA probes known as“molecular beacons” (Tyagi et al., Nat. Biotech., 16: 49-53 (1998)).Molecular beacons have a hairpin structure wherein the quencher dye andreporter dye are in intimate contact with each other at the end of thestem of the hairpin. Upon hybridization with a complementary sequence,the loop of the hairpin structure becomes double stranded and forces thequencher and reporter dye apart, thus generating a fluorescent signal.Tyagi et al. reported use of the non-fluorescent quencher dyes includingthe dabcyl ( 4- {[4-(dimethylamino)phenyl]diazenyl}benzoyl moiety,absorbance max=453 nm) used in combination with fluorescent reporterdyes of widely varying emission wavelength (475-615 nm). At the timethis was surprising since FRET requires significant overlap of theabsorption spectrum of the quencher and of the emission spectrum of thereporter. In case of a dabcyl moiety containing (hereinafter “dabcyl”)quencher and some fluorescent dyes, the spectral overlap was extremelylow, yet quenching efficiency was high. Therefore it was proposed thatthe mechanism of quenching for the hairpin form of the beacons was notFRET, but collisional quenching. In fact, the UV spectra of the quencherchanges in the hairpin form of the beacon, providing evidence of themolecular contact and thus of collisional quenching. A related detectionmethod uses hairpin primers as the fluorogenic probe (Nazarenko et al.,Nucl. Acid Res., 25: 2516-2521 (1997)).

[0008] The second format for “real-time” PCR uses DNA probes which arereferred to as “5′-nuclease probes” (Lee et al., Nucl. Acid Res., 21:3761-3766 (1993)). These fluorogenic probes are typically prepared withthe quencher at the 3′ terminus of a single DNA strand and thefluorophore at the 5′ terminus. During each PCR cycle, the 5′-nucleaseactivity of Taq DNA polymerase cleaves the DNA strand, therebyseparating the fluorophore from the quencher and releasing thefluorescent signal. The 5′-nuclease assay requires that the probe behybridized to the template strand during the primer extension step(60-65° C.). They also disclose the simultaneous “real-time” detectionof more than one polynucleotide sequence in the same assay, using morethan one fluorophore/quencher pair. The 5′-nuclease PCR assay isdepicted in FIG. 1.

[0009] Initially it was believed that 5′-nuclease probes had to beprepared with the quencher (usually tetramethylrhodamine (TAMRA))positioned at an internal nucleotide in close proximity to the5′-fluorophore (usually fluorescein (FAM) or tetrachlorofluorescein(TET)) to get efficient FRET. Later it was found that this is notnecessary, and the quencher and the fluorophore can be located at the 3′and 5′ end of the ODN, respectively. It has been proposed that therandom coil structures formed by these fluorogenic probes in solutionallow a 3′-quencher dye to pass within the Forster radius of the5′-fluorophore during the excited state of the molecule.

[0010] A number of donor/acceptor pairs have previously been described,important to the present invention is dabcyl that is used for instanceas a quencher of dansyl sulphonamide in chemosensors (Rothman & Still(1999) Med. Chem. Lett. 22, 509 - 512).

[0011] Surprisingly, there have been no published reports on the use ofdabcyl in 5′-nuclease probes or other FRET probes that use longwavelength fluorophores. As mentioned above, dabcyl was used in thebeacon-type probes but this is a different quenching mechanism whereinthe dabcyl and fluorophore are in intimate contact (collisionalquenching). Dabcyl was used in fluorogenic peptides as a quencher forthe fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonicacid) which emits at short (490 nm blue) wavelength (Matayoshi et al.Science 247: 954-958 (1990)). EDANS also has a lower extinctioncoefficient than dabcyl so it is not surprising that fluorescentquenching was efficient. It was found for the first time in the presentinvention that dabcyl can be used to quench fluorescein in a FRET typemechanism.

[0012] In addition to the 5′-nuclease PCR assay, other formats have beendeveloped that use the FRET mechanism. For example, single-strandedsignal primers have been modified by linkage to two dyes to form adonor/acceptor dye pair in such a way that fluorescence of the first dyeis quenched by the second dye. This signal primer contains a restrictionsite (U.S. Pat. No. 5,846,726) that allows the appropriate restrictionenzyme to nick the primer when hybridized to a target. This cleavageseparates the two dyes and a change in fluorescence is observed due to adecrease in quenching. Non-nucleotide linking reagents to coupleoligonucleotides to ligands have also been described (U.S. Pat. No.5,696,251).

[0013] FRET systems also have applications in enzymology. Proteasecleavable substrates have been developed where donor/acceptor dye pairsare designed into the substrate. Enzymatic cleavage of the substrateseparates the donor/acceptor pair and a change in fluorescence isobserved due to a decrease in quenching. Cleavable donor/acceptorsubstrates have been developed for chymotrypsin (Li et al. Bioconj.Chem., 10: 241-245 (1999)), aminopeptidase P (Hawthorne et al., Anal.Biochem., 253: 13-17 (1997)), stromelysin (Bickett et al., Ann. N. Y.Acad. Sci., 732: 351-355 (1994)) and leukotriene D₄ hydrolase (White etal., Anal. Biochem., 268: 245-251 (1999)). A chemosensor was describedwhere binding of the ligand separates the donor/acceptor pair (Rothmanet al. Biorg. Med. Chem. Lett., 9: 509-512 (1999)).

[0014] In U.S. Pat. No. 5,801,155 it was disclosed that oligonucleotides(ODNs) having a covalently attached minor groove binder (MGB) are moresequence specific for their complementary targets than unmodifiedoligonucleotides. In addition the MGB-ODNs show substantial increase inhybrid stability with complementary DNA target strands when compared tounmodified oligonucleotides, allowing hybridization with shorteroligonucleotides.

[0015] Reagents for fluorescent labeling of oligonucleotides arecritical for efficient application of the FRET assays described above.Other applications such as DNA micro arrays also use fluorescentlylabeled DNA probes or primers, and there is a need for improved reagentswhich facilitate synthesis of fluorescent DNA. In general,phosphoramidite reagents and solid supports are widely used on ODNsynthesis. However, in the state of the art there are not manycommercially available phosphoramidite reagents for introducingfluorescent groups into ODNs.

[0016] Linker groups to attach different ligand groups to ODNs play animportant role in the synthesis of oligonucleotide conjugates. A methodfor the synthesis of 3′-aminohexyl-tailed oligonucleotides (Petrie etal., Bioconj. Chem., 3: 85-87 (1992)), the use of a trifunctionaltrans-4-hydroxy-L-prolinol group (Reed et al., Bioconjug. Chem., 2:217-225 (1991)), diglycolic acid (Pon et al., Nucl. Acids. Res., 25:3629-3635 (1997)), 1,3-diol reagents (U.S. Pat. No. 5,942,610 and U.S.Pat. No. 5,451,463) and the non-nucleotide trifunctional reagent (U.S.Pat. No. 5,696,251) have been reported.

[0017] Resorufin and coumarin derivatives have been extensively used asenzyme substrates to differentiate isozymes of cytochrome P450 (Hauglandet al., Handbook of Fluorescent Probes and Research Chemicals, SixEdition, Eugene, Ore. pp. 235-236. 1996.). Reactive resorufin analogshave been disclosed in U.S. Pat. No. 5,304,645. Activated esters ofcoumarin derivatives are known in the art (Hirshberg et al, Biochem.,37: 10391-5 (1998)). Coumarin-labeled dUTP incorporated in probes wereused for in situ hybridizations (Wiegant et al., Cytogenet. Cell Genet.,63: 73-76 (1993)). Phosphoramidites to introduce labels intooligonucleotides have been described in U.S. Pat. Nos. 5,328,824 and5,824,796.

[0018] Many current hybridization applications, require more than onereporter molecule. In addition although reporter fluorophores areavailable to be used in reporter/quencher pairs, most suffer from havingsome undesirable characteristic, mixtures difficult to separate,positively charged, difficult to synthesize, unstable duringoligonucleotide synthesis or having overlapping emission wavelengthswith other desirable reporters. The present invention provides reagentsfor oligonucleotide probes that address these unfavorablecharacteristics and overcome some or all of the difficulties.

SUMMARY OF THE INVENTION

[0019] The present invention provides quencher molecules based on the4-[4-nitrophenyl)diazinyl]phenylamine and/or the4-[4-nitrophenyl)diazinyl]-naphthylamine structure. The quenchermolecules have improved UV spectral overlap not only with commonly usedfluorescent reporter groups that emit short wavelength range (about 400to 500 nm), but have extended the range to the mid (525 nm=green) tolong (670 nm=red) wavelengths. The quencher chromophores of the presentinvention are non-fluorescent, easily incorporated into DNA synthesisreagents, stable during automated DNA synthesis and during storage andcompatible with no adverse effects on hybridization properties.Moreover, improved signal to noise ratios are observed with thefluorescent reporter dyes over a more extended wavelength range. Thusthe present invention offers considerable advantages over the use ofdabcyl (Nazerenko et al, Nucl. Acids Res., 25: 2516-21 (1997)) as aquenching dye, as used in the prior art.

[0020] In accordance with one aspect of the present invention thequenchers based on the 4-[4-nitrophenyl)diazinyl]phenylamine (and/or the4-[4-nitrophenyl)diazinyl]naphthylamine structure) are modified withlinker structures that allow their easy incorporation into fluorogenicDNA probes during automated DNA synthesis. The invention includessynthesis of phosphoramidites derived from the novel quencher moleculesfor incorporation of the quencher moieties into oligonucleotides duringautomated synthesis, and also synthesis of reagents derived from thenovel quencher molecules for post solid-phase support attachment toamino-tailed oligonucleotides. In a related aspect, the novel quenchermolecules are introduced into oligonucleotides usingpyrazolo-[5,4-d]pyrimidines and pyrimidines phosphoramidites containingthe quenchers attached at the 3′- and 5′-positions, respectively.

[0021] In accordance with another aspect of the invention, threedifferent fluorescent reagent types that are compatible with DNAsynthesis are synthesized or selected and converted into phosphoramiditereagents suitable for incorporation onto ODNs. Specifically, violetfluorescent dyes based on the10-phenyl-1,3,5,7,9,10-hexahydropyrimidino[5′,4′-5,6]pyridino[2,3-d]pyrimidine-2,4,6,8-tetraone(PPT) structure, red fluorescent dyes based on 7-hydroxyphenoxazin-3-one(resorufin) and blue fluorescent dyes based on the structure of coumarinare incorporated into phosphoramidite reagents. These fluorescent dyeshave excellent properties for multicolor fluorescent analysis incombination with other dyes (eg. fluorescein). These reagents arevaluable for a variety of analytical methods that use either directdetection of fluorescence or FRET detection formats. In a related aspectof the invention the PPT-, coumarin- and resorufin-based fluorophores(fluorescent dyes) are converted into novel reagents suitable for“post-oligonucleotide-synthesis” covalent attachment at the 5′-end ofODNs. In another aspect, the new fluorescent dyes are incorporated intooligonucleotides using pyrazolo-[5,4-d]pyrimidines and pyrimidinesphosphoramidites which contain the fluorophores attached at the 3- and5-positions, respectively.

[0022] In accordance with still another aspect of the invention, ODNscovalently linked with the novel quencher structures of the invention,paired with a covalently attached fluorescent moieties, are prepared.The resulting FL-ODN-Q conjugate may also include a minor groove binder(MGB) that improves the binding and discrimination characteristics ofthe resulting FL-ODN-Q-MGB conjugate in diagnostic assays, particularlyin the TaqMan PCR assay of single nucleotide polymorphism (and the like)where allele-specific discrimination not only requires probes withdifferent fluorescent reporter molecules but efficient quenchers. Thequenchers used in accordance with the invention in the FL-ODN-Q-MGBconjugates provide broad quenching wavelength range, and certain novelreporter labeling reagents in accordance with the invention havedistinctive emission wavelengths for improved multicolor analysis.

[0023] In one application of the principles summarized above,fluorogenic probes are prepared using a universal “3′-hexanol” solidsupport (available in accordance with Gamper et al. Nucleic Acids Res.,21: 145-150 (1993) expressly incorporated herein by reference), where aquencher phosphoramidite of the invention is added at the first couplingstep (3′-end) of the ODN sequence and a fluorophore (FL) was attached atthe final coupling step, yielding 5′-FL-ODN-Q-hexanol conjugate probes.

[0024] As other applications of the invention, methods for synthesizingand attaching the novel quenchers to ODN-fluorophore conjugates with andwithout a 3′-minor groove binder (MGB) are disclosed. These methodsutilize synthetic solid supports for automated oligonucleotide synthesiswith cleavable linkers.

[0025] In another application, a fluorogenic oligonucleotide probe isprepared from a MGB modified solid support substantially in accordancewith the procedure of Lukhtanov et al. Bioconjugate Chem., 7: 564-567(1996), where a quencher-phosphoramidite of the invention is added atthe first coupling step to the MGB, and a fluorophore (FL) is attachedat the final coupling step to the ODN, to yield 5′-FL-ODN-Q-MGBconjugate probe.

[0026] Additional application of the methods and compositions of thepresent invention are in micro-arrays in nucleic acid-based diagnosticassays which recently have become important in many fields, such as themedical sciences, forensics, agriculture and water quality control.Other related application of the methods and compositions of the presentinvention are in procedures using arrays of oligonucleotides, such asthe array-based analysis of gene expression (Eisen, Methods of Enzym.,303: 179-205 (1999)). In these procedures, an ordered array ofoligonucleotides or DNAs that correspond to all, or a large fraction ofthe genes in many organism is used as a platform for hybridization.Microarray-based methods are used in assays to measure the relativerepresentation of expressed RNA species. The quantitation of differencesin abundance of each RNA species is achieved by directly comparing twosamples by labeling them with spectrally distinct fluorescent dyes andmixing the two probes for simultaneous hybridization to one array.

[0027] To the extent the application of the compositions and methods ofpresent invention relates to the detection of nucleic acids, it includesbut is not limited to methods where FRET is involved, such as5′-nuclease, universal energy transfer primers or beacon assays. Thesemethods are usually directed to, but are not limited to the detection ofPCR-generated nucleic acid sequences. Some of these methods involvesimultaneous detection of more than one nucleic acid sequence in thesame assay. Similarly, the invention relates to methods where FRET isinvolved in the detection of protein concentration or enzyme activity.

[0028] Still other applications of the invention relate to the labelingwith luminescent PPT- , coumarin- and resorufin-based dyes of nucleicacids, proteins and other materials including, drugs, toxins, cells,microbial materials, particles, glass or polymeric surfaces and thelike, at a reactive group such as an amino, hydroxyl or sulfhydrylgroup. The present invention may be used in single- and two-steplabeling processes. In the two-step labeling process, a primarycomponent, such as an oligonucleotide is labeled with the reagentcapable of introducing the novel fluorophore PPT-, coumarin- andresorufin-based dyes, by reaction with a reactive group of the ODN (such as an amine, hydroxyl, carboxyl, aldehyde or sulfhydryl group) andthe label is used to probe for a secondary component, such as anoligonucleotide target.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic representation of real-time 5′-nuclease PCRassay.

[0030]FIG. 2 is a graph showing the UV spectra of Dabcyl- and Red13dye-modified DNA probes.

[0031]FIG. 3 is a graph showing the performance of fluorogenic MGBprobes in a “real-time” PCR assay.

[0032]FIG. 4 is a graph showing the fluorescent spectra of violet, FAMand resorufin dye containing DNA probes.

DETAILED DESCRIPTION OF INVENTION QUENCHER REAGENTS FOR OLIGONUCLEOTIDESYNTHESIS

[0033] Two types of reagents for introducing the substituted4-(phenyldiazenyl)phenylamine quencher moieties into oligonucleotidesusing an automated DNA synthesizer are exemplified in the disclosurebelow. Here and in the reaction schemes the abbreviations MGB, FL, Q,CPG and ODN stand for “minor groove binder”, “fluorescent orfluorophor”, “quencher” “controlled pore glass” and “oligonucleotide”moieties or molecules, respectively, and in a manner which is apparentfrom context.

[0034] Dimethoxytrityl Protected Quencher Phosphoramidites

[0035] The first type of reagents disclosed herein are phosphoramiditesthat bear the quencher molecule (Q) as well as a dimethoxytrityl (DMTr)(methoxytrityl, trityl or the like acid labile blocking group) protectedprimary alcohol that provides an attachment point for the growingoligodeoxynucleotide (ODN) chain during subsequent oligonucleotidesynthesis. Examples of these reagents are depicted in Formulas 1, 2, and3, and in Reaction Schemes 1 and 2.

[0036] In Reaction Scheme 1 the starting compound is a substituted4-(phenyldiazenyl)phenylamine 1 that has a primary hydroxyl group. Suchstarting material is commercially available or can be synthesized inaccordance with methods known in the art, applying routine skillavailable to the practicing organic chemist. For example4-nitrobenzendiazonium salt is reacted with 2-(2-chloroanilino)ethanolto yield 2-[2-chloro-4-(4-nitrophenylazo)anilino]ethanol in accordancewith the teachings of U.S. Pat. No. 2,264,303.2-[2-chloro-4-(4-nitrophenylazo)anilino]ethanol is within the scope ofcompound 1 as depicted in Reaction Scheme 1. The specification of U.S.Pat. No. 2,264,303 is expressly incorporated herein by reference.

[0037] Other examples of commercially available starting materials (orof their precursors) are: 2-(ethyl{4-[(4nitrophenyl)diazenyl]phenyl}amino)-ethan-1-ol and2-(ethyl{4-[(2-methoxy-4-nitrophenyl)diazenyl]phenyl}-amino)ethan-1-ol.

[0038] As is shown in Reaction Scheme 1, compound 1 is reacted withp-nitrophenylchloroformate to yield the carbonate 2. Reaction of 2 withsubstituted pyrrolidinediols yields a diol intermediate 3. Thepyrrolidinediol is a trifunctional reagent that has an amino, a primaryand a secondary hydroxyl group. An example of a pyrrolidinediol as wellas examples of other trifunctional reagents having an amino, primary anda secondary hydroxyl group, are described in U.S. Pat. No. 5,512,667 thespecification of which is incorporated herein by reference. The diol-3is reacted first with dimethoxytrityl chloride (DMTrCl) to block theprimary hydroxyl group of the trifunctional reagent and yieldintermediate 4. The intermediate 4, still having a free secondaryhydroxyl group in the trifunctional reagent, is then reacted with2-cyanoethyl diisopropylchlorophosphoramidite to give thedimethoxytrityl protected phosphoramidite reagent 5. In the compoundsshown in Reaction Scheme 1 the symbols are defined as follows. R₀, R₁,R₂, R₃ and R4 are independently —H, halogen,—O(CH₂)_(n)CH₃,—(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃, —N[(CH₂)_(n′)CH₃]₂ where n′=0to 5 or —CN; R₅=—H or —(CH₂)_(n′)CH₃ where n″=0 to 5; R₆=—(CH₂)_(n*)where n_(*)=1 to 5 and q=1 to 20. The dimethoxytrityl protectedphosphoramidite reagent 5 is suitable for attachment to oligonucleotidesin steps otherwise known in routine ODN synthesis.

[0039] In other embodiments, using the reactions described in ReactionScheme 1, (or only such modifications which are within the skill of thepracticing organic chemist) starting with other trifunctional reagentshaving an amino and two hydroxyl groups, the phosphoramidites of Formula1 and Formula 2 are synthesized, where q, R₀, R₁, R₂, R₃, R₄ and R₅, aredefined as above; r and s independently are 1 to 20; X is —O— or —CH₂—;t and v independently are 1 to 20.

[0040] Reaction Scheme 2 discloses the synthesis of another exemplaryphosphoramidite reagent 10 bearing the substituted4-(phenyldiazenyl)-phenylamine quencher moiety and including thetrifunctional pyrrolidinediol moiety. In this synthetic scheme thestarting material is a substituted 4-(phenyldiazenyl)phenylaminecompound 6 that has a free carboxyl group. Compound 6 (commerciallyavailable or made in accordance with the chemical literature within theskill of the practicing organic chemist) is reacted withpentafluorophenyl trifluoroacetate to make an active ester 7, which isthereafter reacted to couple the substituted4-(phenyldiazenyl)-phenylamine moiety to the ring nitrogen of apyrrolidinediol moiety having a free primary and a free secondaryhydroxyl group, yielding compound 8. Treatment of 8 with DMTrCl followedby reaction with 2-cyanoethyl diisopropylchlorophosphoramidite gives thedimethoxytrityl protected phosphoramidite reagent 10. In Reaction Scheme2 the symbols are defined the same as in Reaction Scheme 1.

[0041] In still another example, using the reactions described inReaction Scheme 2, starting with a substituted4-(phenyldiazenyl)phenylamine (compound 6) and using a non-cyclictrifunctional reagent (having an amino and two hydroxyl functions)instead of the pyrrolidinediol shown in Scheme 2, the dimethoxytritylprotected phosphoramidite of Formula 3 is synthesized, where q, R₀, R₁,R₂, R₃, R₄, R₅, and R₆ are defined as above and t and v independentlyare 1 to 20.

[0042] Quenchers Attached to Solid Support Through (or Similarly)Protected Linker, Suitable for ODN Synthesis

[0043] A second class of compounds or reagents suitable for introducingthe quencher molecules into ODNs constitute a composition that has asolid support of the type used for ODN synthesis (for example controlledpore glass (CPG)), and linker attaching the quencher to the solidsupport. The linker has a hydroxyl function that is protected, usuallyby a dimethoxytrityl group which is removed during the synthesis whenthe first nucleotide is attached to the linker. Generally speaking thesame quencher/linker intermediates described above in Reaction Scheme 1can also be used to prepare these reagents (CPG beads) having theexemplary structure 12, shown in Reaction Scheme 3.

[0044] In accordance with this scheme, the secondary hydroxyl group ofthe intermediate 4 (shown in Scheme 1) is reacted with succinicanhydride, and thereafter pentafluorophenyl trifluoroacetate to providethe active ester 11; The active ester 11 is then reacted with the freeamino group attached to the. solid support (CPG bead) to provide themodified solid support 12. Whereas the exemplary modified solid support12 includes the “trifunctional linker” derived from pyrrolidine diol, itwill be readily understood by those skilled in art that analogousmodified solid supports including other linkers and related structures,such as the linkers shown in Formulas 1, 2 and 3 can also be madesubstantially in accordance with Reaction Scheme 3, resulting inmodified solid support compositions including the quencher moiety, suchthe ones shown in Formula 4 and Formula 5.

[0045] The modified solid support compositions including the quenchermoiety of structure 12 and of Formula 4 and 5 are used for preparing3′-quencher conjugates, allowing the introduction of a fluorophore atthe 5′-end with the appropriate phosphoramidite, or post-syntheticallywith a fluorophore containing a reactive group. In Reaction Scheme 3 andin Formula 4 and Formula 5 the symbols are defined as above. It shouldbe understood that other solid supports (such as polystyrene) and othercleavable linker systems (in addition to the succinate linker shown) canalso be prepared in accordance with these generic teachings andtherefore are also within the scope of the invention.

[0046] Minor Groove Binder Quencher Reagents for OligonucleotideSynthesis

[0047] In one embodiment a minor groove binder (MGB) is attached tocontrolled pore glass (CPG) through a cleavable linker. A quenchermoiety, based on the 4-(phenyldiazenyl)phenylamine structure, isattached through a linker molecule to the MGB. The linker molecule alsocontains a hydroxyl group blocked with DMTr (or like) blocking group.After removal of the DMTr group, an oligonucleotide is synthesized on anautomated oligonucleotide synthesizer by step-wise attachment ofnucleotide units to the hydroxyl group. A fluorophore is introduced atthe 5′-end with the appropriate phosphoramidite, or post-syntheticallywith a fluorophore containing a reactive group, to yield an ODN havingan attached fluorescent moiety (FL), quencher (Q) and MGB(FL-ODN-Q-MGB). In this connection it is noted that the synthesis ofMGBs and their attachment to ODNs is well known (see for example U.S.Pat. No. 5,801,155, 09/539,097 and 09/141,764; all of which areexpressly incorporated herein by reference.

[0048] In a preferred embodiment the MGB is3-{[3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[3,2-e]indoline-7-carboxylicacid (DPI₃). The synthesis of the covalently bound “aggregate”FL-ODN-Q-DPI₃ requires five phases, described below. The first phase,shown in Reaction Scheme 4, is the synthesis of an intermediate,2-(4-nitrophenyl)ethyl3-(pyrrolo[4,5-e]indoline-7-carbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(DPI₂-NPC) 17. The second phase, shown in Reaction Scheme 5, is thesynthesis of Q-DMTr-DPI-CO₂PFP 24 where a quencher is coupled through alinker to a pyrrolo[3,2-e]indoline-7-carboxylic acid unit (DPI ). Here,and in the reaction schemes PFP stands for the pentafluorophenyl orpentafluorophenyloxy group, as the context requires. In the third phase,shown in Reaction Scheme 6, DMTr-Q-DPI₃-PFP 25a is synthesized from 17and 24. In the fourth phase 25a is coupled to CPG to yield a DMTr-Q-DPI₃-CPG 29, and in the fifth phase 29 is used on an automatedoligonucleotide synthesizer to stepwise attach nucleotide units and toprovide, after removal from the CPG, the product FL-5′-ODN-3′-Q-DPI₃ 30.

[0049] The fourth and fifth phases of these synthetic process are shownin Reaction Scheme 7. Experimental conditions for this sequence (phases1 through 5) are described below.

[0050] Describing these phases or reactions now in more detail, Q-DPI₃moiety 25 (phase 3) is synthesized by the reaction of two intermediates,17 and 24 as shown in Reaction Scheme 6. The first intermediate DPI₂-NPE17 is made as shown in Scheme 4. DPI-tBoc 13 was reacted withp-nitrophenylethanol in the presence of diethylazodicarboxylate (DEAD)and triphenylphosphine to yield the di-ester 14. Compound 14 was firsttreated with trifluoroacetic acid (TFA) to yield 15, and then conjugatedwith 13 in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride to give the 4-nitrophenylester of DPI₂ 16 in good yield. Reaction of 16 with TFA gives thep-nitrophenethyl ester of DPI₂ 17. The second intermediate DPI-Q 24(phase 2) is synthesized as shown in Reaction Scheme 5. A substitutednitroaniline 18 (available commercially or in accordance with thechemical literature) is diazotized in the presence of nitrous acid andis coupled to a substituted aniline 19 (available commercially or inaccordance with the chemical literature) to form the azo intermediatequencher molecule 20. Alkaline hydrolysis of the ethyl ester 20 followedby la the treatment with DMTrCl gives the DMTr-Q 21, that issubsequently activated with pentafluorophenyl trifluoroacetate to yield22. Reaction of 22 with DPI-methyl ester gives the Q-DMTr-DPImethylester 23. Compound 23 is then treated with alkali to hydrolyze themethyl ester and then activated with PFP-TFA to yield Q-DMTr-DPIPFPester 24. In Reaction Scheme 5 the symbols R₀, R₁ through R₄, v and tare defined as above.

[0051] Referring now to Reaction Scheme 6, where the symbols are alsodefined as above, DMTr-Q-DPI₃-PFP 25a (third phase) is synthesized firstby reacting the activated quencher 24 (DMTr-Q-DPI PFP) with DPI₂-NPC 17to yield the p-nitrophenylethyl ester 25, which is converted to theactive ester 25a, first by treatment with base such as1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to remove thep-nitrophenylethyl moiety and then treatment with2,3,4,5,6-pentafluorophenyl trifluoroacetate (PFP-TFA).

[0052] The synthesis of DMTr-Q-DPI₃-CPG 29 (phase four) is shown inReaction Scheme 7. In this synthetic sequence the improved quenchermolecule becomes attached through a cleavable diglycolate linker tocontrolled pore glass beads (CPG). Specifically, aminopropanol, or ahomolog thereof, is reacted successively with monomethoxytrityl chloride(MMTr-Cl) and then with diglycolic anhydride to form MMT-blockedaminopropanol 26 (or homolog) and MMT-diglycolate 27, respectively. Thesymbol m is defined as an integer having the values 2 to 20. For thepresently preferred aminopropanol m is 3. The remaining symbols in thisscheme are defined as above. Reaction of 27 with long chain aminoalkylCPG in the presence of activating agents (HOBT and HBTU), yields theMMT-diglycolate-CPG 28, that is converted after detritylation andreaction with 25a to DMTrO-Q-DPI₃-CPG 29.

[0053] In phase 5, still shown in Reaction Scheme 7 oligonucleotidesynthesis is performed with the aid of an automated DNA synthesizer, anda fluorophore is attached at the 5′-end of the ODN, using either afluorphore-phosphoramidite or a fluorophore containing a reactive group,to yield the FL-ODN-Q-DPI₃ 30 conjugate.

[0054] The FL-ODN-Q-DPI₃ 30 conjugate can also be synthesized by analternative synthetic route which is not specifically illustrated in thereaction schemes. In this alternative route DPI₃-methyl ester (obtainedin accordance with Boger et al., J. Org. Chem., 52: 1521-(1987)incorporated herein by reference) is first reacted with compound 22 andthen with alkali to give Q-DPI₃-methyl ester and Q-DPI₃-COOH,respectively. The latter compound is then activated withpentafluorophenyl trifluoroacetate, to yield 25a, which is then used inthe reactions shown in Scheme 7, to yield 30.

[0055] FL-ODN-O and FL-ODN-O-MGB Probes

[0056] A general structure of a preferred embodiment of Fl-ODN-Q-DPI₃conjugates is shown in Formula 6 where:

[0057] FL is a fluorophore with emission wavelengths in the range ofabout 300 to about 800 nm and more preferably 400 to 700 nm; K is alinker containing between 1 and 30 atoms, which include any of C, O, N,S, P and H; [A-B]_(n) symbolizes a DNA, RNA or PNA or any combinationthereof, where A is the sugar phosphate backbone (including modifiedsugars and modified phosphates), B is the heterocyclic base, and n isthe number of nucleotide units. B can independently be any of thepurine- and pyrimidine-; pyrazolo[3,4-d]pyrimidine-, 7-substitutedpyrazolo[3,4-d]pyrimidine- , 7-deazapurines, 7-substiuted7-deazapurines, modified purine- and pyrimidine-bases, and theoligonucleotide or nucleic acid can include any combinations of thesebases. W is a linker of the length of 0 to approximately 30 atoms,selected from the group consisting of C, O, N, S, P and H, furthermore Wis a substituted branched aliphatic chain, or a substituted ringstructure or a combined substituted aliphatic and ring structure; R₀,R₁, R₂, R₃ and R₄ are as described previously and m=1 to 20.

[0058] Syntheses of PNA and PNA/DNA chimeras are known in the art andcan generally speaking be performed in accordance with the publicationsUhlmann et al., Angew. Chem. Inter. Ed., 37:2796-2823 (1998); Mayfieldet al., Anal. Biochem., 401-404 (1998) which are incorporated herein byreference.

[0059] A still more preferred embodiment within the scope of Formula 6is one where W is —(CH₂)₃N(—)—(CH₂)₃—; R₀=NO₂; R₁=—Cl; R₂=R₃=R₄=H; K isa 6 carbon linker and m=3.

[0060] Conjugate probes of the present invention containing afluorescent reporter-quencher pair are, generally speaking, used inconjunction with the amplification of target polynucleotides, frequentlyin methods utilizing PCR, as described for example by Holland et al.Proc. Acad. Sci., 88: 7276-7280(1991) and Wittwer et al., Biotechniques,22: 176-181 (1997) which are incorporated herein by reference. Thebinding site of the conjugate probe is located between the PCR primersused to amplify the target polynucleotide.

[0061] Use of the conjugate oligonucleotide probes according to thepresent invention for detection of target oligonucleotide sequencesprovides several advantages over prior-art reporter quencher groups andcombinations. For example, the quenchers including the4-[4-nitrophenyl)diazinyl]phenylamine structure in accordance with thepresent invention gave larger signal to noise ratios (S/N) in probeswith either FAM or TAMRA serving as reporters than dabcyl as a quencher.Furthermore, the quenchers in accordance with the invention show abroader absorbance range than dabcyl, allowing efficient quenching of abroad range of fluorophores. In addition, in MGB-oligonucleotideconjugates have improved hybridization characteristics, an improvedquencher showed about 30-fold increase in S/N ratio with TAMRA comparedto a standard probe (no DPI₃) with dabcyl. Reagents of the presentinvention allow the introduction of the quencher during automatedoligonucleotide synthesis. (Dabcyl phosphoramidite is commerciallyavailable; (Glen Research, Sterling, Va.))

[0062] It should be understood that, generally speaking, for the purposeof this invention, an oligonucleotide comprises a plurality ofnucleotide units, a 3′ end and a 5′end. The oligonucleotide may containone or more modified bases other than the normal purine and pyrimidinebases, as well as modified internucleotide linkages capable ofspecifically binding target polynucleotide through Watson-Crick basepairing, or the like. In addition, oligonucleotides may include peptideoligonucleotides (PNAs) or PNA/DNA chimeras, the synthesis of which isknown and can be performed for example in accordance with thepublications Uhlmann et al., Angew. Chem. Inter. Ed., 37:2796-2823(1998) and Mayfield et al., Anal. Biochem., 401-404 (1998); all of whichare expressly incorporated herein by reference.

[0063] Generally, the oligonucleotide probes of the invention will havea sufficient number of phophodiester linkages adjacent to the 5′ end toallow 5′-3′ exonuclease activity to allow efficient cleavage between thequencher and fluorophore molecules in the probe. An adequate number inthis regards is approximately between 1 and 100.

FLUOROPHORE REAGENTS Coumarin Phosphoramidite Reagents

[0064] Fluorescent dyes which have emission wavelengths shorter than thegreen fluorescent dye FAM also have utility in DNA probe based assays.However in the prior art these probes have been less popular sinceexcitation with laser light sources is less feasible than with longerwavelengths. To the best knowledge of the present inventors, to date,there have been no reports of DNA synthesis reagents that contain bluefluorescent dyes. An example of such a phosphoramidite reagentcontaining a preferred coumarin fluorophore and which is suitable forDNA synthesis, is shown in Reaction Scheme 8, as compound 34. In thephosphoramidite reagent 34, R₈ and R₉ independently are H, halogen,—NO₂, —SO₃, —C(═O)NH₂, or —CN;—OR_(nn), —SR_(nn),—OR_(nn),—NHR_(nn),—N[R_(nn)]₂ where R_(nn) is independently H, ablocking group compatible with oligomer synthesis and which can beremoved under acid or alkaline conditions; or a group that containsbetween 1 and 10 carbon atoms, j and k independently are 1 to 10. As canbe seen the reagent 34 includes a covalently linked coumarin chromophorewhich emits light at 458 nm. DNA probes containing this coumarinchromophore were prepared and gave the desired fluorescent emissionproperties.

[0065] Referring now to Scheme 8 in general terms and also in an examplethat provides the specific phosphoramidite reagent 34a, a hydroxylsubstituted (2-oxo-2H-chromen-4-yl)-alkylcarboxyl methyl ester (31) isobtained according to the publication Baker et al. (J.Chem.Soc.; 170,173 (1950)) incorporated herein by reference. Compound 31 is convertedto the alkanol derivative 32 (specifically to 32a where R₈ is —OH and R₉is —H) by reaction with an aminoalkanol at 80° C. Reaction of 32 firstwith DMTrCl and then with trimethylacetic anhydride followed by theremoval of the DMTr blocking group gives a pivaloate derivative 33, inthe specific example 33a where R₈ is —OC(═O)CH(CH₃)₂ and R₉ is —H.Reaction of 33 with 2-cyanoethyl diisopropylchlorophosphoramidite givesreagent 34 (specifically 34a where R₈ is —OC(═O)CH(CH₃)₂, R₉ is —H). Thereagent 34 is used for incorporating the coumarin fluorophore into the5′-terminus of DNA probes. It is noteworthy that removal of theprotecting groups during automated oligonucleotides synthesis proceededwell, resulting in high yields. The symbols j and k in Scheme 8 aredefined as 0 to 20 and 1 to 20, respectively.

[0066] Resorufin Phosphoramidite

[0067] Another new class of DNA synthesis reagents are based on the7-hydroxy-3H-phenoxazin-3-one chromophore present in the parent compound(resorufin) and have emission wavelength (595 nm) that is easilydistinguished from FAM emission. In accordance with the invention thechromophore is synthesized in such a way as to incorporate a linkerstructure for further functionalization to the desired phosphoramiditereagents. The preparation of preferred examples of these reagents 37suitable for DNA synthesis, is shown in Reaction Scheme 9. Generallyspeaking in reagent 37 R₁₀ and R₁₁, independently are H, —OR₁₂, —NHR₁₃,halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂, O-alkyl or O-alkanoyl where the alkyl or alkanoylgroup has 1 to 10 carbons, or —CN where n=0 to 5; h=1 to 20; and R₁₂ andR₁₃ are blocking groups compatible with ODN synthesis. R₁₄ in the schemeis H or DMTr.

[0068] As is shown in the example of Scheme 9 in general terms and alsofor a specific example, reaction of nitrosorecorcinol derivative(commercially available or synthesized in accordance with thestate-of-the-art) and of 4-(3-hydroxypropyl)benzene-1,3-diol (obtainedin accordance with Forchiassin et al. J. Heterocyc. Chem. 20, 1983,493-494.) and MnO₂ yielded a resazurin derivative contaminated with someresorufin derivative. This mixture was treated with NH₄OH and Zn dust toyield resorufin derivative 35 (specifically 35a where R₁₀ is OH, and R₁₁is H) contaminated with 2,3,4-trihydro-2H-pyrano[3,2-b]phenoxazin-9-oneas major impurity. The latter mixture was treated with DMTrCl andpyridine, and then with trimethylacetic acid anhydride. The product 36was then subjected to purification by chromatography on silica gel togive the DMTr-protected derivative of 36a (where R₁₀ is —OC(═O)C(CH₃)₃,R₁₁, is H and R₁₄ is DMTr). The pure DMTr-derivative was treated withTFA/CH₂Cl₂ to yield a single product 36b after silica gelchromatography. Treatment of 36 (where R₁₀ is —OC(═O)C(CH₃)₃, R₁₁, andR₁₄ are H) with 2-cyanoethyl diisopropylchlorophosphoramidite gave thedesired phosphoramidite reagent 37 (specifically 37a) that is utilizedto introduce the fluorophore into a desired ODN.

[0069] PPT Phosphoramidite

[0070] The synthesis of a phosphoramidite reagent incorporating a purplefluorescent dye PPT 44 having excitation and emmision wavelengths of 384and 400 nm, respectively is shown in Reaction Scheme 10 and in ExampleX. In accordance with this scheme 6-chloro-3-n-butyluracil 38 and2-(4-aminophenyl)ethanol 39 are reacted to yield the phenyl substituteduracil derivative 40. The compounds 38 and 39 can be obtained inaccordance with the state-of-the-art and the chemical literature.Reaction of 40 with 5-formyl-4,6-dichloro pyrimidine in DMF at roomtemperature affords the tricyclic heterocycle 41. Reduction of 41 inNH₃/Na₂S₂O₄ yields 42 which is thereafter blocked as thetoluoyl-derivative 43. In the final step 43 is reacted with 2-cyanoethyldiisopropylchlorophosphoramidite to yield the reagent PPT cyanoethylphoporamidite 44 that is used to introduce the PPT fluorophore into anODN.

[0071] In still further embodiments, the fluorophores coumarin,resorufin and PPT substituted with an alkylcarboxyl group serve asstarting materials for the synthesis of the correspondingphosphoramidite reagents. The fluorophores coumarin, resorufin and PPTsubstituted with an alkylcarboxyl group are either commerciallyavailable or can be synthesized in accordance with the state-of-the-art.These compounds are activated on the alkylcarboxyl group as thepentafluorophenyl esters. The activated esters are used to attach thesedyes to amine modified oligonucleotides.

[0072] Similarly, in still other embodiments, dUTP-labeled quenchers orfluorophores are obtained for example in accordance with the teachingsof U.S. Pat. No. 5,328,824). Furthermore, the phosphoramidite of7-labeled pyrazolo[3,4-d]pyrimide-labeled quenchers or fluorophores aresynthesized according to the teaching of 5,824,796 (incorporated hereinby reference) and can be used for labeling of oligonucleotides.

[0073] PPG Red Dye-based and Other Phosphoramidite Reagents forOligonucleotide Synthesis.

[0074] In another embodiment the red dye 13 quencher is attached to the3-position of pyrazolo[5,4-d]pyrimidines (PP) or the 5-position of apyrimidine. Referring now to Scheme 11 itself, the starting material is5-(4-amino-3-iodopyrazolo[5,4-d]pyrimidinyl)-2-(hydroxymethyl)oxolan-3-ol45 which is available in accordance with the publication Seela et al. J.Chem. Soc., Perkin. Trans., 1 (1999, 479-488) incorporated herein byreference. Compound 45 is first reacted withN-propynyl-2,2,2-trifluoroacetate (or a homolog thereof where in thescheme n is 1 to 10) and then with Pd(PPh₃)₄-CuI to give the alkynederivative 46. Pd/H₂ reduction of 46 followed by ammonium hydroxidetreatment gives the aminoalkyl derivative 47 (PPA′). Reaction of PPA′with compound 2 (available as disclosed in connection with ReactionScheme 1) yielded substituted PPA′-Red 13 48. Reaction of 48 with(1,1-dimethoxyethyl)dimethylamine blocks the amino group of thepyrazolo[5,4-d]pyrimidine to yield 49. 49 is first reacted with DMTrCland then with 2-cyanoethyl diisopropylchlorophosphoramidite to give theDMTrCl blocked PPA′-Red 13 phosphoramidite 50.

[0075] In still other embodiments starting with the deoxyriboside of6-amino-5-hydroxy-3-iodo-pyrazolo[5,4-d]pyrimidin-4-one (3-Iodo-PPG) thephosphoramidite reagent containing the Red 13 dye covalently linked tothe 3-Iodo-PPG moiety is synthesized with reactions analogous to thoseshown in Reaction Scheme 11. Similarly starting with5-aminopropyldeoxyuridine the phosphoramidite reagent containing the Red13 dye covalently linked to 5-aminopropyl-deoxyuridine is synthesized.

[0076] It will be clear to those skilled in the art in light of theforegoing disclosure that the pyrazolopyrimidine-Red-13- or uridine-Red13-based phosphoramidites within the scope of this invention may containvarious linkers between the pyrazolopyrimidine and uracil bases and theRed 13 quenchers, to the full extent such linkers are available inaccordance with the state of the art and this disclosure.

EXAMPLES

[0077] The methods and compositions of the present invention can be usedwith a variety of techniques, both currently in use and to be developed,in which hybridization of an oligonucleotide to another nucleic acid isinvolved. These include, but are not limited to, techniques in whichhybridization of an oligonucleotide to a target nucleic acid is theendpoint; techniques in which hybridization of one or moreoligonucleotides to a target nucleic acid precedes one or morepolymerase-mediated elongation steps which use the oligonucleotide as aprimer and the target nucleic acid as a template; techniques in whichhybridization of an oligonucleotide to a target nucleic acid is used toblock extension of another primer; techniques in which hybridization ofan oligonucleotide to a target nucleic acid is followed by hydrolysis ofthe oligonucleotide to release an attached label; and techniques inwhich two or more oligonucleotides are hybridized to a target nucleicacid and interactions between the multiple oligonucleotides aremeasured. The conditions for hybridization of oligonucleotides, and thefactors which influence the degree and specificity of hybridization,such as temperature, ionic strength and solvent composition, arewell-known to those of skill in the art. See, for example, Sambrook etal., supra; Ausubel et al., supra; Innis et al. (eds.) PCR Protocols,Academic Press, San Diego, 1990; Hames et al. (eds.) Nucleic AcidHybridisation: A Practical Approach, IRL Press, Oxford, 1985; and vanNess et al. (1991) Nucleic Acids Res. 19:5143-5151.

[0078] Hybridization Probes

[0079] In one application of the present invention, one or moreFL-oligonucleotide conjugates are used as probe(s) to identify a targetnucleic acid by assaying hybridization between the probe(s) and thetarget nucleic acid. A probe may be labeled with any detectable label ofthe present invention, or it may have the capacity to become labeledeither before or after hybridization, such as by containing a reactivegroup capable of association with a label or by being capable ofhybridizing to a secondary labeled probe, either before or afterhybridization to the target. As a basis of this technique it is notedthat conditions for hybridization of nucleic acid probes are well-knownto those of skill in the art. See, for example, Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold SpringHarbor Laboratory Press (1989); Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons (1987, 1988, 1989, 1990, 1991,1992, 1993, 1994, 1995, 1996); Hames et al. (eds.) Nucleic AcidHybridization: A Practical Approach, IRL Press, Oxford, 1985; and vanNess et al. Nucleic Acids Res. 19:5143-5151(1991).

[0080] Hybridization can be assayed (i.e., hybridized nucleic acids canbe identified) by distinguishing hybridized probe from free probe by oneof several methods that are well-known to those of skill in the art.These include, but are not limited to, attachment of target nucleic acidto a solid support, either directly or indirectly (by hybridization to asecond, support-bound probe or interaction between surface-bound andprobe-conjugated ligands) followed by direct or indirect hybridizationwith probe, and washing to remove unhybridized probe; determination ofnuclease resistance; buoyant density determination; affinity methodsspecific for nucleic acid duplexes (e.g., hydroxyapatitechromatography); interactions between multiple probes hybridized to thesame target nucleic acid; and other known techniques. See, for example,Falkow et al., U.S. Pat. No. 4,358,535; Urdea et al., U.S. Pat. Nos.4,868,105 and 5,124,246; Freifelder, Physical Biochemistry, SecondEdition, Freeman & Co., San Francisco, 1982; Sambrook, et al., supra;Ausubel et al., supra; and Hames et al., supra.

[0081] Assays Utilizing Labeled Probes, Hydrolyzable Probe and LabeledPrimers

[0082] Additional applications for oligonucleotide conjugates containinga fluorophore and quencher are found in assays in which a labeled probeis hybridized to a target and/or an extension product of a target, and achange in the physical state of the label is effected as a consequenceof hybridization. A probe is a nucleic acid molecule that is capable ofhybridizing to a target sequence in a second nucleic acid molecule. Byway of example, one assay of this type, the hydrolyzable probe assay,takes advantage of the fact that many polymerizing enzymes, such as DNApolymerases, possess intrinsic 5′-3′ exonucleolytic activities.Accordingly, if a probe is hybridized to a sequence that can serve as atemplate for polymerization (for instance, if a probe is hybridized to aregion of DNA located between two amplification primers, during thecourse of an amplification reaction), a polymerizing enzyme that hasinitiated polymerization at an upstream amplification primer is capableof exonucleolytically digesting the probe. Any label attached to such aprobe will be released, if the probe is hybridized to its target and ifamplification is occurring across the region to which the probe ishybridized. Released label is separated from labeled probe and detectedby methods well-known to those of skill in the art, depending on thenature of the label. For example, radioactively labeled fragments can beseparated by thin-layer chromatography and detected by autoradiography;while fluorescently-labeled fragments can be detected by irradiation atthe appropriate excitation wavelengths with observation at theappropriate emission wavelengths. This basic technique is described forexample in U.S. Pat. No. 5,210,015 incorporated herein by reference.

[0083] In a variation of this technique, a probe contains both afluorescent label and a quenching agent, which quenches the fluorescenceemission of the fluorescent label. In this case, the fluorescent labelis not detectable until its spatial relationship to the quenching agenthas been altered, for example by exonucleolytic release of thefluorescent label from the probe. Thus, prior to hybridization to itstarget sequence, the dual fluorophore/quencher labeled probe does notemit fluorescence. Subsequent to hybridization of thefluorophore/quencher-labeled probe to its target, it becomes a substratefor the exonucleolytic activity of a polymerizing enzyme which hasinitiated polymerization at an upstream primer. Exonucleolyticdegradation of the probe releases the fluorescent label from the probe,and hence from the vicinity of the quenching agent, allowing detectionof a fluorescent signal upon irradiation at the appropriate excitationwavelengths. This method has the advantage that released label does nothave to be separated from intact probe. Multiplex approaches utilizemultiple probes, each of which is complementary to a different targetsequence and carries a distinguishable label, allowing the assay ofseveral target sequences simultaneously.

[0084] The use of FL-ODN-Q-DPI₃ conjugates in this and related methodsallows greater speed, sensitivity and discriminatory power to be appliedto these assays. In particular, the enhanced ability ofMGB-oligonucleotide conjugates to allow discrimination between a perfecthybrid and a hybrid containing a single-base mismatch facilitates theuse of hydrolyzable probe assays in the identification ofsingle-nucleotide polymorphisms and the like, as described in thepublication WO 995162A2, incorporated herein by reference. Examples 13and 14 illustrate the utility of FL-ODN-Q-DPI₃ conjugates in this typeof assay. It will be clear to those of skill in the art thatcompositions and methods, such as those of the invention, that arecapable of discriminating single-nucleotide mismatches will also becapable of discriminating between sequences that have multiplemismatches with respect to one another.

[0085] Another application embodiment uses a self-probing primer with anintegral tail, where the quencher/fluorophore is present in the hairpin,that can probe the extension product of the primer and afteramplification hybridizes to the amplicon in a form that fluoresces. Theprobing of a target sequence can thereby be converted into aunimolecular event (Whitcombe, D. et al., Nat. Biotech., 17: 804-807(1999)).

[0086] Fluorescence Energy Transfer

[0087] In further applications of the novel compositions of thisinvention, oligonucleotide conjugates containing a fluorophore/quencherpair (FL-ODN-Q) are used in various techniques which involve multiplefluorescent-labeled probes. In some of these assays changes inproperties of a fluorescent label are used to monitor hybridization. Forexample, fluorescence resonance energy transfer (FRET) has been used asan indicator of oligonucleotide hybridization. In one embodiment of thistechnique, two probes are used, each containing a fluorescent label anda quencher molecule respectively. The fluorescent label is a donor, andthe quencher is an acceptor, wherein the emission wavelengths of thedonor overlap the absorption wavelengths of the acceptor. The sequencesof the probes are selected so that they hybridize to adjacent regions ofa target nucleic acid, thereby bringing the fluorescence donor and theacceptor into close proximity, if target is present. In the presence oftarget nucleic acid, irradiation at wavelengths corresponding to theabsorption wavelengths of the fluorescence donor will result in emissionfrom the fluorescence acceptor. These types of assays have the advantagethat they are homogeneous assays, providing a positive signal withoutthe necessity of removing unreacted probe. For further details andadditional examples of these assays which are per se known in the art,see, for example, European Patent Publication 070685; and thepublication Cardullo et al. (1988) Proc. Natl. Acad. Sci. USA 85:8790-8794, both of which are incorporated herein by reference.Additional applications of the novel compositions of the presentinvention are in those and related techniques in which interactionsbetween two different oligonucleotides that are both hybridized to thesame target nucleic acid are, measured. The selection of appropriatefluorescence donor/fluorescence acceptor pairs will be apparent to oneof skill in the art, based on the principle that, for a given pair, theemission wavelengths of the fluorescence donor will overlap theabsorption wavelengths of the acceptor. The enhanced ability ofDPI₃-oligonucleotide conjugates to distinguish perfect hybrids fromhybrids containing a single base mismatch facilitates the use ofFRET-based techniques in the identification of single-nucleotidepolymorphisms and the like.

[0088] In another application of the novel compositions of theinvention, the fluorescence of the FL-ODN-Q conjugate is quenched in itsnative state. But, after hybridization with the intended target thespatial arrangement of the fluorophore and quencher moieties are changedsuch that fluorescence occurs. For this basic technique see for exampleTyagi et al., Nat. Biotech., 16: 49-53 (1998); and U.S. Pat. No.5,876,930, both of which are incorporated herein by reference.

[0089] It should be understood that in addition to the fluorophoreswhich are found in accordance with the present invention especiallyuseful to be used with the quenchers of the invention, and whichfluorophores are incorporated into ODNs in accordance with theinvention, a person of ordinary skill may choose additional fluorophoresto be used in combination with the quenchers of the present invention,based on the optical properties described in the literature, such as thereferences: Haugland Handbook of Fluorescent Probes and ResearchChemicals, Six Edition, Eugene, Ore. pp. 235-236. 1996; Berlman,Handbook of Fluorescence Spectra of Aromatic Molecules, 2^(nd),Accademic Press, New York, 1971; Du et al., PhotochemCAD. AComputer-Aided Design and Research Tool in Photochemistry, Photochem.Photobiol. 68, 141-142 (1998). Therefore the use of the novel ODNquencher conjugates in combination with these known fluorophores isconsidered within the scope of the invention.

[0090] In another application, the minor groove binder, DPI₃, is coupledto a quencher in a FL-ODN-Q-CDPI₃ conjugate to improve signal to noiseratios (See Table 2). Preferred quenchers are the quenchers of Formula 6and more preferred the quenchers are 8-11, 12-16 and 30.

[0091] Additional quenchers suitable for use in combination with thenovel fluorophores (34, 37 and 44) of the invention includedabcylnitrothiazole, TAMRA,6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid,6-carboxy-X-rhodamine (Rox) and QSY-7.

[0092] Another application of the novel fluorophore/quencher pairs ofthe invention is to incorporate the pair into enzyme substrates, wherefluorescence is quenched because of the proximity of the fluorophore andquencher. However, after an enzyme cleaves the substrate the fluorophoreand quencher become separated and fluorescence is observed. An exampleof this technique is described below using the phosphodiesterase enzyme.It will be clear to those schooled in the art that suitable substratescontaining both the novel quenchers and fluorophores can be constructedfor enzymes that cleave substrates.

[0093] Oligonucleotide Arrays

[0094] In another application, FL-ODNs of the present invention areutilized in procedures employing arrays of oligonucleotides. Examplesfor this technique that is per se known in the art include sequencing byhybridization and array-based analysis of gene expression. In theseprocedures, an ordered array of oligonucleotides of different knownsequences is used as a platform for hybridization to one or more testpolynucleotides, nucleic acids or nucleic acid populations.Determination of the oligonucleotides which are hybridized and alignmentof their known sequences allows reconstruction of the sequence of thetest polynucleotide. For a description of these techniques see forexample, U.S. Pat. Nos. 5,492,806; 5,525,464; 5,556,752; and PCTPublications WO 92/10588 and WO 96/17957 all of which are incorporatedherein by reference. Materials for construction of arrays include, butare not limited to, nitrocellulose, glass, silicon wafers and opticalfibers.

STRUCTURAL CONSIDERATIONS

[0095] The terms oligonucleotide, polynucleotide and nucleic acid areused interchangeably to refer to single- or double-stranded polymers ofDNA or RNA (or both) including polymers containing modified ornon-naturally-occurring nucleotides, or to any other type of polymercapable of stable base-pairing to DNA or RNA including, but not limitedto, peptide nucleic acids which are disclosed by Nielsen et al. (1991)Science 254:1497-1500; bicyclo DNA oligomers (Bolli et al. (1996)Nucleic Acids Res. 24:4660-4667) and related structures. One or more MGBmoieties and/or one or more fluorescent labels, and quenching agents canbe attached at the 5′ end, the 3′end or in-an internal portion of theoligomer. A preferred MGB in accordance with the invention is DPI₃ andthe preferred quencher is red 13 amide.

[0096] Preferred in the present invention are DNA oligonucleotides thatare single-stranded and have a length of 100 nucleotides or less, morepreferably 50 nucleotides or less, still more preferably 30 nucleotidesor less and most preferably 20 nucleotides or less with a lower limitbeing approximately 5 nucleotides.

[0097] Oligonucleotide conjugates containing a fluorophore/quencher pairwith or without an MGB may also comprise one or more modified bases, inaddition to the naturally-occurring bases adenine, cytosine, guanine,thymine and uracil. Modified bases are considered to be those thatdiffer from the naturally-occurring bases by addition or deletion of oneor more functional groups, differences in the heterocyclic ringstructure (i e., substitution of carbon for a heteroatom, or viceversa), and/or attachment of one or more linker arm structures to thebase. The modified nucleotides which may be included in the ODNconjugates of the invention include 7-deazapurines and their derivativesand pyrazolopyrimidines (described in PCT WO 90/14353 incorporatedherein by reference); and in co-owned and co-pending application Ser.No. 09/054,630.

[0098] Preferred base analogues of this type include the guanineanalogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG or PPG) andthe adenine analogue 4-amino-1H-pyrazolo[3,4d]pyrimidine (ppA or PPA).Also of use is the xanthine analogue1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione (ppX). These baseanalogues, when present in an oligonucleotide, strengthen hybridizationand improve mismatch discrimination. All tautomeric forms ofnaturally-occurring bases, modified bases and base analogues may beincluded in the oligonucleotide conjugates of the invention.

[0099] Similarly, modified sugars or sugar analogues can be present inone or more of the nucleotide subunits of an oligonucleotide conjugatein accordance with the invention. Sugar modifications include, but arenot limited to, attachment of substituents to the 2′, 3′ and/or 4′carbon atom of the sugar, different epimeric forms of the sugar,differences in the α- or β- configuration of the glycosidic bond, andother anomeric changes. Sugar moieties include, but are not limited to,pentose, deoxypentose, hexose, deoxyhexose, ribose, deoxyribose,glucose, arabinose, pentofuranose, xylose, lyxose, and cyclopentyl.

[0100] Modified internucleotide linkages can also be present inoligonucleotide conjugates of the invention. Such modified linkagesinclude, but are not limited to, peptide, phosphate, phosphodiester,phosphotriester, alkylphosphate, alkanephosphonate, thiophosphate,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, substituted phosphoramidate and the like. Severalfurther modifications of bases, sugars and/or internucleotide linkages,that are compatible with their use in oligonucleotides serving as probesand/or primers, will be apparent to those of skill in the art.

[0101] Certain preferred embodiments of the invention involve thesynthesis of numerous phophoramidites with various quencher chromophoresand linkers and their incorporation at the 3′-end of fluorogenic MGBODNs as shown in Reaction Scheme 3. Different fluorescent reportergroups (shown in Reaction Scheme 7) were also incorporated into theoligonucleotide probes and are described in the EXPERIMENTAL section.The fluorogenic properties of these ODN conjugates are described inTable 2. In other embodiments MGB molecules, due to their desirableimproved hybridization properties, were incorporated intooligonucleotides containing both a fluorophore and a quencher, withoutloss in hybridization specificity, fluorescent quenching and fluoresentsignal. The flat aromatic quencher residue coupled to the neighboringaromatic DPI₃ residue, have strict geometric requirements since thelinker between the oligonucleotide and the DPI₃ residue must be flexibleenough to allow positioning of the DPI₃ in the minor groove after DNAduplex formation.

[0102] Characteristics of Reagents of the Invention

[0103] A number of FL-ODN-Q-DPI₃ conjugates synthesized with thereagents and methods of the invention are shown in Formulas 7 to 16,where n specifies the number of bases in the oligonucleotide and R₂ iseither FAM or TAMRA. “B” signifies a heterocyclic base attached to thedeoxyribose sugar moiety.

Linker Structure R₁ Formula 7

Formula 8 (X = Cl) Formula 9 (X = H)

Formula 10 (p = 1) Formula 11 (p = 3)

Formula 12

Formula 13

Formula 14 (q = 2, X = H) Formula 15 (q = 2, X =Cl) Formula 16 (q = 3, X= Cl)

Formulas 7 to 16

[0104] The quenchers incorporated in the compounds represented byFormulas 7-16 are the commercially available2-[4-(4-nitrophenylazo)-N-ethylphenylamino]ethanol (Disperse Red 1),2-[4-(2-chloro-4-nitrophenylazo)-N-ethylphenylamino]ethanol (DisperseRed 13) and 2-[4-(dimethylamino)phenylazo]benzoic acid, identified inthis invention as Red1, Red 13 and dabcyl respectively.

[0105] UV Properties of Red 13 and Dabcyl Oligonucleotide Conjugates

[0106]FIG. 2 shows the absorbance properties of the red13 chromophore(Formula 8, without DPI₃) in comparison to dabcyl (Formula 7, withoutDPI₃ when incorporated at the 3 ′-end of an otherwise unmodified DNAprobe. The broader absorbance (especially at long wavelengths) of thered13 chromophore is a clear advantage. Note that the 8_(max) for red13is at 522 nm whereas the 8_(max) for dabcyl is 479 nm. The absorbance ofred13 is ideal for quenching of fluorescein (emission max=525 nm) butalso overlaps with the fluorescence emission of other common laser dyes.

[0107] Quenching Properties of DPI₃ Probes with Various Quenchers andLinkers.

[0108] For the 10 fluorogenic probes described in Formulas 7 to 16 thefluorescence of a standard solution of each probe was measured beforeand after digestion with snake venom phosphodiesterase (PDE), asdescribed in the EXPERIMENTAL section. This PDE assay allows thequenching properties of each probe to be compared. Fluorescence of thedigested probe (signal) divided by the initial fluorescence (noise) gavea signal to noise ratio (S/N), presented in Table 1. Larger numbers forS/N reflect more efficient fluorescent quenching (lower fluorescentbackground) of the intact probe. TABLE 1 Effect of different quenchersand linkers on fluorogenic probes shown in Formulas 7-16. Formula #(quencher) S/N^(a) (R₂ = FAM) S/N^(a) (R₂ = TAMRA)  7 (dabcyl) 16 13  8(red13) 21 21  9 (red1) 24 21 10 (red13) 13 33 11 (red13) 27 21 12(dabcyl) 13 7 13 (red13) 23 21 14 (red1) 19 3 15 (red13) 24 4 16 (red13)22 24

[0109] It is clear from the data in Table 1 that the red13 chromophoreand the closely related red1 chromophore are better quenchers for bothFAM and TAMRA with a variety of linkers than dabcyl. The linker canaffect quenching by the red13 chromophore. For example, Formula 14 andFormula 15 worked well with FAM, but had poor quenching efficiency forTAMRA. It is somewhat surprising that dabcyl worked so well, especiallyfor is the TAMRA probes. As described below, effective FRET quenching bydabcyl is a specific case for MGB probes.

Linker Structure R₁ Formula 17

Formula 18 (X = Cl)

Formulas 17 to 18

[0110] Comparison of Quenching Properties of DPI₃ Probes and Probeswithout DPI₃.

[0111] To further show the advantages of the red13 quencher chromophore,fluorogenic probes with a 3′-hexanol blocking group (without MGB) werecompared. The structure and fluorescent properties of 13 fluorogenicprobes with the same sequence were compared using the PDE assay. Ared-sensitive detector was used in this study (Table 2) whereas ablue-sensitive detector was used in the study shown in Table 1 (S/N foridentical ODNs are different because different detectors have differentsensitivities for the same fluorphore). The following structuralvariables are summarized in Table 2: Probe type (no-MGB vs. MGB ),Quencher (dabcyl vs. Red13 vs. Red13 amide), and Reporter dye (FAM vs.TAMRA). TABLE 2 Fluorescent properties of oligonucleotides with variousQuenchers/Fluorophores¹. Formula # Probe type Quencher FAM (S/N) TAMRA(S/N) 17 no-DPI₃ dabcyl 4.7 3.9 18 no-DPI₃ red13 11.6 5.8  7 DPI₃ dabcyl23 23.5  8 DPI₃ red13 35 108 30 (R1 = 2 − DPI₃ red13 amide 48 97 Cl, t =v = 3)

[0112] It is clear from the data shown in Table 2, that in the probeswhich do not contain DPI₃ the dye Red 13 quencher works better thandabcyl for both FAM and TAMRA. In DPI₃ containing probes, the dye Red13works better in combination with FAM and much better in combination withTAMRA. Both 8 and 30 work better in DPI₃ containing probes with bothfluorophores, with is 30 showing the best S/N ratio for FAM Thus, it wasfound that the Red13 chromophore is a more efficient quencher-thandabcyl for long wavelength fluorescent reporter groups. For the mostcommonly used fluorophore (FAM) a 2.5-fold increase in S/N was observedfor standard (no-DPI₃) probes. This improved quenching by red13 isconsistent with the increased spectral overlap presented in FIG. 2 and astandard FRET mechanism. The increased SIN of both 8 and 30 whenincorporated into the DPI₃ probes is dramatic and surprising. Thecombination of the red13 quencher and the DPI₃ resulted in a 10-foldincrease in S/N for FAM quenching and a 28-fold increase in S/N forTAMRA quenching.

[0113] It is surprising and that the DPI₃ residue helps improvefluorescent quenching by the dabcyl and red13 chromophores. Withoutwishing to be bound by theory, it is presently postulated that therandom coil conformation of the fluorogenic probe in solution is morestructured in the DPI₃ probes such that the average distance between thefluorophore and quencher is closer than in probes without MGB. Thiscloser average distance in the DPI₃ probes (tighter coil) would giverise to more efficient FRET. The exact nature of this interaction is notknown, but UV spectra of the quencher and dye chromophores are notaffected by the DPI₃. This is in contrast to the fluorogenic hairpinprobes where the UV spectra are changed by the constrained conformation(collisional quenching).

[0114] Performance of Fluorogenic DPI₃ Probes in a “Real-Time” PCRAssay.

[0115] DPI₃ probes prepared with 5′-fluorescein and the red13 amidelinker were tested in the 5′-nuclease assay to see if the hybridizationproperties were compatible with the linker system. As shown in FIG. 3,both dabcyl and Red13 worked as quenchers for fluorescein in the5′-nuclease assay when used in MGB probes. Red13 performed better thandabcyl as evidenced by the lower initial fluorescence (background) andthe higher plateau after PCR. Current commercially availablethermal-cycling fluorimeters can not read longer wavelength dyes inreal-time PCR, but the red13 chromophore was shown to give a good S/Nwith TAMRA containing probes in an end point analysis after PCR.

[0116] According to another general method, the 5′-fluorophore-ODN-Q-MGBconjugates of the instant invention have improved performance in assaysdesigned to detect DNA targets by direct hybridization. A basicdescription of this method is found in U.S. Pat. No. 5,876,930,incorporated herein by reference. In this assay format, thenon-hybridized probes (quenched by FRET) become fluorescent upon forminga rigid duplex structure, thereby separating the quencher andfluorophore. Red13 Chromophore Quenches a Broad Range of FluorescentReporter Groups

[0117] A series of DPI₃ probes with the red13 amide were prepared withseveral different fluorescent reporter groups to examine the effectiverange of quenching. Probes were digested with PDE as usual and showedgood S/N for dyes which emit from 458 -665 nm. TABLE 3 Performance ofFluorogenic DPI₃ Probes with Various Fluorophores. Fluorophore (FL) Ex 8(nm) Em 8 (nm) S/N coumarin 378 458 32 FAM 488 522 63 Cy3 541 565 61TAMRA 547 582 37 resorufin 549 595 110 Cy5 641 665 36

[0118] The structure of the fluorogenic probes was FL-ODN-Q-CDPI₃ whereQ is the red13 amide and the ODN sequence was 5′-GTC CTG ATT TTA C(SEQUENCE Id. No.2). The fluorophores FAM, TAMRA, cy3 and cy5 wereincorporated using commercially available phosphoramidite reagents. Thecoumarin and resorufin fluororophores were incorporated usingphosphoramidites 34 and 37 which were prepared as described below.

[0119] The fluorescent emission is well separated from FAM, as shown inthe overlayed spectra in FIG. 4. As shown in Table 3, the resorufinfluorescence is also quenched by the red13 chromophore. Thus theresorufin phosphoramidite has excellent properties for use in FRETprobes and in combination with FAM for multicolor analysis.

[0120] As shown in Table 3, the coumarin fluorescence is also quenchedby the red13 chromophore. Thus, the coumarin phosphoramidite reagent canbe incorporated in FRET probes and particularly in combination with FAMfor multicolor analysis.

[0121] Fret-based Enzyme Substrates

[0122] The improved quencher molecules can be used in other FRET basedassay systems. According to another general application of theinvention, a quencher molecule and fluorophore are attached to an enzymesubstrate, which through its catalytic action on thisQ-substrate-fluorophore conjugates cleaves and separates the Q andfluorophore molecules. For example, the pentafluorophenyl activatedester 11 shown in Reaction Scheme 3 can be used for labeling lysineresidues of peptides for studying proteolytic enzymes.

EXPERIMENTAL PROCEDURES

[0123] General Experimental

[0124] All air and water sensitive reactions were carried out under aslight positive pressure of argon. Anhydrous solvents were obtained fromAldrich (Milwaukee, Wis.). Flash chromatography was performed on 230-400mesh silica gel. Melting points were determined on a Mel-Temp meltingpoint, apparatus in open capillary and are uncorrected. Elementalanalysis was performed by Quantitative Technologies Inc. (Boundbrook,N.J.). TV-visible absorption spectra were recorded in the 200-400-nmrange on a UV-2100 (Shimadzu) or a Lambda 2 (Perkin Elmer)spectrophotometers. ¹H NMR spectra were run at 20EC on a Bruker WP-200or on a Varian XL-200 spectrophotometer; chemical shifts are reported inppm downfield from Me₄Si. Thin-layer chromatography was run on silicagel 60 F-254 (EM Reagents) aluminum-backed plates.

Example 1 2-({4-[(2-Chloro4-nitrophenyl)diazenyl]phenyl}ethylamino)ethyl(2S,4R)-2-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}-4-{[bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}pyrrolidinecarboxylate(5).

[0125] 2-({4-[(2-Chloro-4-nitrophenyl)diazenyl]phenyl}ethylamino)ethyl(5S, 3R)-3-hydroxy-5-(hydroxymethyl)pyrrolidinecarboxylate (3).

[0126] A solution of2-[4-(2-chloro-4-nitrophenylazo)-N-ethylphenylamino]ethanol (DisperseRed 13, Aldrich Chemical Co., 9.0 g, 25.80 mmol) and 4-nitrophenylchloroformate (Aldrich Chemical Co., 9.4 g, 46.61 mmol) in 90 ml ofanhydrous pyridine was stirred at 70° C. for 40 min, affordingintermediate 2. Ethanol (5.0 ml) was added to the reaction solutionfollowed by trans-hydroxyprolinol (Reed et al. Bioorg. Chem. 2: 217-225(1991) (42 ml of a 0.5 M solution in ethanol) and triethylamine (3.2ml). The resultant solution was stirred for 30 min at 70 ° C. Thesolution was evaporated to dryness and the residue was suspended in 1liter of water and extracted with ethyl acetate (3×500 ml). The pooledextracts were dried over sodium sulfate, filtered and evaporated. Theresidue was purified by silica gel chromatography eluting with agradient of 0-10% methanol in ethyl acetate. The pure product fractionswere evaporated and precipitated from ethyl acetate-ether: 9.2 g (59%);TLC (ethyl acetate), R_(f)=0.25. ¹H NMR (DMSO-d₆) δ8.43 (1H, d, J=2.5Hz), 8.25 (1H, dd, J=9.0 and 2.4 Hz), 7.86 (2H, d, J=9.1 Hz), 7.78 (1H,d, J=9.0 Hz), 6.96 (2H, d, J=9.3 Hz), 4.88 (1H, m), 4.67 (1H, t, J=5.7Hz), 4.19 (3H, m), 3.80 (1H, m), 3.73 (2H, t, J=5.4 Hz), 3.56 (2H, q),3.46 (1H, t, J=4.7 Hz), 3.27 (1H, m), 1.94 (1H, m), 1.79 (1H, m), 1.17(3H, t, J=6.8 Hz). Anal. Calcd for C₂₂H₂₆CIN₅O₆+0.2 H₂O: C, 53.32; H,5.37; N, 14.13. Found: C, 53.24; H, 5.25; N, 13.99.

[0127] 2-({4-[(2-Chloro-4-nitrophenyl)diazenyl]phenyl}ethylamino)ethyl(5S,3R)-5-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}-3-hydroxypyrrolidinecarboxylate(4).

[0128] To a solution of 3 (9.1 g, 18.53 mmol) in 130 ml of anhydrouspyridine was added 6.26 g of dimethoxytrityl chloride. The solution wasstirred for 3 h. at room temperature and then poured into 300 ml of 5%sodium bicarbonate solution. The mixture was extracted with ethylacetate (2×300 ml) and the combined extracts were dried over sodiumsulfate, filtered and evaporated. The residue was purified by silica gelchromatography eluting with a gradient of 20-0% hexane in ethyl acetatefollowed by a gradient of 0-2% methanol in ethyl acetate. Thechromatography eluent also contained 1% triethylamine. The pure productfractions were combined affording an amorphous solid: 12.66 g (86%); TLC(ethyl acetate), R_(f)=0.44. ¹H NMR (DMSO-d₆) δ8.45 (1H, s), 8.26 (1H,d, J=8.9 Hz), 7.82 (3H, m), 7.27 (4H, m), 7.16 (5H, m), 6.95-6.79 (6H,m), 4.95 (1H, m), 4.32 (1H, m), 4.14 (1H, m), 3.99 (2H, m), 3.73 (1H,m), 3.69 (6H, s), 3.56 (1H, m), 3.40-3.30 (2H, m), 3.14 (1H, m),2.10-1.82 (2H, m), 1.16 (3H, m), 1.06 (3H, t, J=6.5 Hz). Anal. Calcd forC₄₃H₄₄CIN₅O₈+0.2 H₂O: C, 64.73; H, 5.61; N, 8.78. Found: C, 65.08; H,5.70; N, 8.31.

[0129] 2-({4-[(2-Chloro-4-nitrophenyl)diazenyl]pheny}ethylamino)ethyl(2S,4R)-2-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}-4-{[bis(methylethyl)amino](2-cyano-ethoxy)phosphinooxy}pyrrolidinecarboxylate(5).

[0130] To a solution of 4 (12.63 g, 15.91 mmol) dissolved in 440 ml ofanhydrous methylene chloride, containing 8.0 ml ofN,N-diisopropylethylamine, was added 5.94 ml of 2-cyanoethyldiisopropylchlorophosphoramidite. The solution was stirred 30 min underargon at room temperature. The reaction mixture was treated with 10 mlof methanol and poured into 400 ml of 5% sodium bicarbonate solution.The organic phase was dried over sodium sulfate and evaporated. Theresidue was purified by silica gel chromatography eluting with agradient of 40-20% hexane in ethyl acetate (2% triethylamine). The pureproduct fractions were evaporated affording an amorphous solid: 14.75 g(93% yield). ³¹P NMR (DMSO-d₆) δ146.93 (singlet). Anal. Calcd forC₅₂H₆₁CIN₇O₉+1.0 H2O: C, 61.68; H, 6.27; N, 9.68. Found: C, 61.44; H,6.47; N, 9.35.

Example 2 RED 13-pyrrolidine-DMTr-CPG 12

[0131] Synthesis of pentafluorophenyl ester (11) and RED13_pyrrolidine_DMTr_CPG (12) Reactions Scheme 3.

[0132] The pentafluorophenyl ester (11) is synthesized by the samemethod used for the synthesis of Compound 22 as described in Example 4and Reaction Scheme 5.

[0133] RED 13-pyrrolidine-DMTr-CPG (12)

[0134] 10 g of LCAA_CPG was combined with 5 ml of a 0.3 M solution of 11in DMF and agitated gently overnight, when it was filtered and washedwith 2×100 mL of DMF, 2×100 mL of acetonitrile, and 2×100 mL of ether.Traces of ether were removed in vacuo (oil pump). Unreacted amino groupswere acetylated by treating the CPG with 40 mL of dry pyridine and 5 mLof acetic anhydride. After swirling for 1.5 h, the CPG was filtered andwashed with 2×100 mL of DMF, 2×100 mL of acetonitrile, and 2×100 mL ofether. Traces of ether were removed in vacuo (oil pump). The CPG wasanalyzed for MMT loading by treating 3_5 mg of CPG in 25 mL of 1:1/70%perchloric acid:methanol. The absorbance of the released MMT cation wasrecorded at 472 nm and loading level was adjusted to be between 30_(—)40mmol/g of CPG using the equation:

MMT loading (mmol/g)=A472×volume (in mL)×14.3, wt of CPG (mg)

Example 3 2-(4-Nitrophenyl)ethyl3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(17). (Reaction Scheme 4)

[0135] 2-(4-Nitrophenyl)ethyl3-[(tert-butyl)oxycarbonyl]pyrrolo[4,5-e]indoline- 7-carboxylate (14).

[0136] Ten grams (33.1 mmol) of3-[(tert-butyl)oxycarbonyl]pyrrolo[3,2-e]indoline-7-carboxylic acid(Boger, D. L., Coleman, R. S., Invergo, B. J. (1987) J Org. Chem. 52,1521.), well dried, are placed into an argon filled flask, and 84 mL ofTHF and 10.4 mL (66.2 mmol) of diethyl azodicarboxylate (DEAD) areadded. Then a dropping funnel is placed atop the flask (flushed withargon) and a water bath (to cool the flask) is placed under it. Asolution of 17.3 g (66 mmol) of triphenylphosphine and 6.64 g (39.7mmol) of 2-(p-nitrophenyl) ethanol in 160 mL of ethyl ether is made.This solution is added to the dropping funnel, and then to the reactionflask, dropwise, with stirring. The reaction is allowed to proceed foran hour, at which time, a TLC analysis is done (2:1 hexanes/ethylacetate) examined by UV (254 nm) to determine whether the reaction iscomplete. If it is complete, then the baseline spot (bluish) willdisappear and the product, with an R_(f) of 0.55, will appear as a darkspot. Often, especially if the reactants are not entirely dry, anotherportion of triphenylphosphine and DEAD are required. If so, a tenth ofthe original amounts is usually sufficient, i.e., 1.73 g oftriphenylphosphine and 1.04 mL of DEAD. These can be added neat to thestirred solution. Allow to react another hour, after which another TLCanalysis usually reveals complete reaction. The product usuallyprecipitates out in part; this is collected bt filtration and washedwith methanol, then recrystallized by dissolving in a minimum amount(typically, 80-100 ml) of warm acetone and adding four times that volumeof warm methanol. Cool to 4° C. for several hours or perhaps overnight.The supernatant from the original precipitation is saved and evaporatedto a syrup or until dry. It too is dissolved in a minimum amount of warmacetone; typically about 100 - 120 mL of warm acetone. The total amountor acetone used for the two recrystallizations is usually approximately200 mL. As before, an amount of warm methanol equal to four times theamount of acetone is added. The solution is cooled; crystallizationbegins almost at once but is allowed to. continue several hours toovernight. The recrystallizations are quite efficient, but the productfrom the reaction supernatant is usually not quite as pure and ispurified by recrystallization. The yield is approximately 85%. (mp191-193° C.) ¹H NMR (DMSO-d6) δ11.83 (s, 1H), 8.18 (d, J=8.5 Hz, 2H),7.84 (br s, 1H), 7.64 (d, J=8.5 Hz, 2H), 7.25 (d, J=8.8 Hz), 6.96 (s,1H), 4.56 (t, J=6 Hz, 2H), 4.00 (t, J=8.8 Hz, 2H), 3.21 (m, 4H), 1.51(s, 9H). Combustion Analysis: Found: C, 63.16%; H, 5.56%; N, 9.45%.Calculated for 0.4 mole added water: C, 62.8%; H, 5.7%; N, 9.16%.

[0137] 2-(4-Nitrophenyl)ethyl pyrrolo[4, 5-e]indoline-7-carboxylate(15).

[0138] Two grams (4.43 mmol) of 14 are weighed into a round bottomedflask. Then, in a fume hood, 25 mL (325 mmol) of trifluroacetic acid isadded, and the flask is capped and stirred. The solid dissolves in abouta minute. The mixture is stirred for 1 hour, at which time deprotectionwill be done (HPLC can be used as a check). The acid is evaporated on arotary evaporator (use a trap) and the product is dissolved in 100 mL ofmethylene chloride. This is extracted twice with 100 mL of half to ⅔saturated sodium bicarbonate solution. The aqueous layers areback-extracted once with ˜50 mL of methylene chloride and this iscombined with the rest. The organic layer is dried over sodium sulfatetwice and evaporated to give a brown solid. If desired, this materialcan be recrystallized by diluting a very concentrated solution inmethylene chloride with methanol and cooling. Yields approaching 100%are usually obtained. mp 192-194° C. ¹H NMR (DMSO-d6) δ 11.51 (s, 1H),8.18 (d, J=8.5 Hz, 2H), 7.63 (d, J=8.5 Hz, 2H), 7.11 (d, J=8.5 1H), 6.80(s, 1H), 6.70 (d, J=8.5 Hz), 5.03 (br s, 1H), 4.54 (t, J=6.4 Hz, 2H),3.46 (t, J=8.6 Hz, 2H), 3.19 (m, 2H), 3.04 (t, J=8.6 Hz, 2H). CombustionAnalysis: Calculated for C₁₉H₁₇N₃O₄: C,64.94%; H, 4.88%; N, 11.96%.Found: C, 65.50%; H 4.70%; N, 11.64%

[0139] 2-(4-Nitrophenyl)ethyl3-({3-[(tert-butyl)oxycarbonyl]pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(16).

[0140] 3.09 grams (8.8 mmol) of 15 is mixed with 2.66 grams (8.8 mmol)of 13 (Boger, D. L., Coleman, R. S., Invergo, B. J. (1987) J. Org. Chem.52, 1521.), and 46 mL of DMF is added. Then 3.85 grams (8.77 mmol) of1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride areadded. The mixture is stirred for about three hours. The mixture isinitially homogeneous, but as the stirring proceeds, a precipitate ofthe product forms. The solvent DMF is evaporated under a high vacuum,and about 100 mL of methanol is added. The mixture is swirled andfiltered in a sintered glass funnel, then thoroughly washed with 3×50 mLportions of methanol. Then it is dried in vacuo. Yields usually approach100 percent. mp: 132E-134° C. ¹H NMR (DMSO-d6)*: 11.93 (s, NH, 1H),11.62 (s, NH, 1H), 8.30 (br s, aromatic proton, 1H), 8.27 (br s,aromatic proton, 1H), 8.19 (d, aromatic protons, J=8.3 Hz, 2H), 7.65 (d,aromatic protons, J=8.3 Hz, 2H), 7.34 (d, J=9 Hz, aromatic proton, 1H),7.29 (d, J=9 Hz, aromatic proton, 1H), 7.07 (s, aromatic proton, 1H),6.98 (s, aromatic proton, 1H), 4.60 (m, aliphatic protons, 4H), 4.02 (t,J=8.5 Hz, aliphatic protons, 2H), 3.40 (t, J=8 Hz, aliphatic protons,2H), 3.24 (m, aliphatic protons, 4H), 1.52 (s, 3×CH₃, 9H). CombustionAnalysis: Calculated: C, 66.13%; H, 5.23%; N, 11.02%. Found: C, 65.94%;H, 5.19%; N, 11.07%.

[0141] 2-(4-Nitrophenyl)ethyl3-(pyrrolo[4,5-e]indolin-7-ylcarbonyl)pyrrolo[4,5-e]indoline-7-carboxylate(17).

[0142] 5 grams of 16 are placed in a flask. 100 mL of trifluoroaceticacid is added, and the mixture is stirred. After an hour, the acid isevaporated on a rotary evaporator and 100 mL saturated sodiumbicarbonate solution and 100 mL of water are added. The mixture isagitated or sonicated for ˜½ hours, then filtered and washed with waterand then methanol, and dried in vacuo. The material may berecrystallized. It is dissolved in a minimum amount of warm DMF, andthen approximately a threefold portion of methanol is added and thesolution is sonicated a few minutes. A cream to brown materialcrystallizes out. This is washed with methanol, and dried in vacuo. Theyield approaches theoretical values.¹H NMR (DMSO-d6)δ 11.96 (s, NH, 1H),11.71 (s, NH, 1H), 8.30 (br s, aromatic proton, 1H), 8.27 (br s,aromatic proton, 1H), 8.19 (d, aromatic protons, J=8.5 Hz, 2H), 7.66 (d,aromatic protons, J=8.3 Hz, 2H), 7.34 (m, aromatic protons, 2H), 7.08(s, aromatic proton, 1H), 7.03 (s, aromatic proton, 1H), 4.60 (m,aliphatic protons, 4H), 3.68 (t, J=8 Hz, aliphatic protons, 2H), 3.40(t, J=8 Hz, aliphatic protons, 2H), 3.24 (m, aliphatic protons, 4H).Combustion Analysis: Found: C, 63.55 %; H, 4.42 %; N, 11.95 %.Calculated, for ½ mole sodium bicarbonate contaminant: C, 63.43%; H,4.45%; N, 12.13%.

Example 4 2,3,4,5,6-pentafluorophenyl3-[4-({3-[bis(4-methoxyphenyl)phenylmethoxy]propyl}-{4-[(2-chloro4-nitrophenyl)diazenyl]phenyl}amino)butanoyl]pyrrolo[4,5-e]indoline-7-carboxylate(24) (Reaction Scheme 5)

[0143] Ethyl 4-[(3-hydroxypropyl)phenylamino]butanoate (19)

[0144] A mixture of 3-(phenylamino)propan-1-ol (Huang, Yande; Arif, AttaM.; Bentrude, Weseley G.; J.Org.Chem.; 58(23) 1993; 6235-6246) (65.6 g,0.43 mol), ethyl 4-bromobutyrate (104.5 g, 0.54 mol) and 100 mL ofethyldiisopropylamine is stirred at 100° C. for 1 h. The reaction iscooled to room temperature and partitioned between water 400 mL andethyl acetate (500 mL). The organic layer is washed with saturatedNaHCO₃, brine and dried over Na₂SO₄. The oil obtained afterconcentration is chromatographed on silica eluting with 10% EtOH/CHCl₃.Concentration of the appropriate fractions affords 115 g (100%) of thedesired product as a colorless, viscous oil. ¹H NMR (CDCl₃),δ7.23 (m,2H), 6.72 (m, 3H), 4.14 (q, J=7 Hz, 2H), 3.72 (t, J=6 Hz, 2H), 3.43 (t,7 Hz, 2H), 3.34 (t, 7 Hz, 2H), 2.35 (t, 7 Hz), 1.88 (m, 4H), 1.26 (t, 7Hz, 3H).

[0145] Ethyl4-({4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}(3-hydroxypropyl)amino)butanoate(20)

[0146] 2-Chloro-4-nitroaniline 2.5 g (10 mmol) is placed into a 125 mLflask and 6 mL of water is added. Agitation and sonication partiallydissolves the yellow chloronitroaniline. Then the stirred solution iscooled with ice in a fume hood and 15.8 mL of concentrated (˜12 M) HClis added. Most of the yellow material dissolves at this point. The flaskis fitted with a dropping funnel, and a solution of 1.51 g (21.9 mmol)sodium nitrite in 3-4 mL of water is added to the dropping funnel andslowly added to the solution in the flask with stirring, over about 20minutes. When this is complete, 0.6 g (˜21 mmol) of urea is addedfollowed by 2.73 g of ethyl 4-[(3-hydroxypropyl)phenylamino]butanoate asa solution in 8.2 mL acetic acid. After a minute 20 g of sodium acetatein ˜50 mL of water is added. The mixture is allowed to stir for an hourat room temperature. Most of the s product is separated as an emulsion.The mixture is partitioned between ethyl acetate and water. The organiclayer is washed with NaHCO₃ (3×50 ml), brine and dried over anhydroussodium sulfate. Then the organic solvents are evaporated to a syrup. Thecrude product is chromatographed on silica gel (1.5×20 inches) elutingwith 50% ethyl acetate/hexane. The appropriate fractions are collected,combined, evaporated (30-40 degrees), and dried in a vacuum. The productis a dark oil. The yield is approximately 68-70%. ¹H NMR (DMSO-d6) δ8.42(d, J=2.5 Hz, aromatic proton, 1H), 8.24 (dd, J₁=9 Hz, J₂=2.5 Hz,aromatic proton, 1H), 7.86 (d, J=9Hz, 2H), 7.77 (d, J=9 Hz, 1H), 6.92(d, J=9 Hz, aromatic protons, 2H), 4.67 (t, J=6 Hz, OH, 1H), 4.07 (q,J=7 Hz, CH₂O, 2H), 3.5 (m, aliphatic protons, 6H), 2.40 (t, J=7 Hz,aliphatic protons, 2H), 1.84 (m, aliphatic protons, 2H), 1.72 (m,aliphatic protons, 2H), 1.18 (t, J=7 Hz, CH₃, 3H).

[0147]4-({4-[(2-Chloro-4-nitrophenyl)diazenyl]phenyl}(3-hydroxypropyl)amino)butanoicacid.

[0148] To a stirred solution of 20 (4.48 g, 10 mmole) in 40 mL of THFadded 40 mL of ethanol followed by a solution of KOH (0.84 g,1 5 mmol)in 20 mL of water and 20 mL of ethanol. The mixture is stirred overnightand concentrated. The residue suspended in 125 mL of water, treated with2.6 mL (˜3 eqv.) of acetic acid, and cooled to 4° C. The resulting solidis filtered off, washed with water, and dried. Yield is quantitative. ¹HNMR (DMSO-d6) δ 8.42 (d, J=2.5 Hz, aromatic proton, 1H), 8.23 (dd, J₁=9Hz, J₂=2.5 Hz, aromatic proton, 1H), 7.82 (d, J=9Hz, 2H), 7.90 (d,J=9Hz, 1H), 7.03 (d, J=9 Hz, aromatic protons, 2H), 4.8 (br s, OH, 1H),3.5 (m, aliphatic protons, 6H), 1.86 (t, J=6 Hz, aliphatic protons, 2H),1.72 (m, aliphatic protons, 4H).

[0149]4-({3-[Bis(4-methoxyphenyl)phenylmethoxy]propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}amino)butanoicacid (21)

[0150] 4.21 g (10 mmol) of the acid from the previous step is placedinto a 250 mL round bottom flask. Dry pyridine (50-100 ml) is added andevaporated (30-40 degrees) with a rotary evaporator. The process isrepeated once or twice to remove all traces of water. Dry pyridine (80mL) is added to the contents of the flask. Then 4.07 g (12 mmol) ofdimethoxytrityl chloride is added. After being stirred for 1 h pyridineis evaporated and the resulting syrup is dissolved in a few millilitersof 18:1:1 methylene chloride/methanol/triethylamine. A silica gel column(˜1.5″×20″) is prepared with an eluent of 18:1:1 methylenechloride/methanol/ triethylamine and the product is run through thecolumn, collecting and combining the appropriate fractions. After thesolvents are removed by evaporation the resulting amorphous solidcontains some triethylammonium salts in addition to the desired product.The impurity does not interfere with the next step and the product isused without additional purification.

[0151] 2,3,4,5,6-Pentafluorophenyl4-({3-[bis(4-methoxyphenyl)phenylmethoxy]-propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenylamino}butanoate(22).

[0152] To the flask containing 21 (10 mmol) is added 7 mL oftriethylamine followed by 100 mL of methylene chloride, 2.05 mL ofpentafluorophenyl trifluoroacetate (PFP-TFA) is then added. The solutionis stirred for half an hour. At the end of this time, the reaction isusually complete. (TLC: 2:1 hexane/ethyl acetate). The solvent isremoved on the rotary evaporator to give a syrup which ischromatographed on silica eluting with 1:3 ethyl acetate/hexane.Appropriate fractions are collected, combined, evaporated and driedunder vacuum. The yield is 41%. ¹H NMR (DMSO-d6) δ 8.43 (d, J=2.5 Hz,aromatic proton, 1H), 8.24 (dd, J₁=9 Hz, J₂=2.5 Hz, aromatic proton,1H), 7.83 (d, J=9 Hz, aromatic proton, 1H), 7.78 (d, J=9 Hz, aromaticproton, 1H), 7.42-7.15 (m, aromatic protons, 10H), 7.07 (m, aromaticprotons, 2H), 7.00-6.80 (m, aromatic protons, 4H), 3.72 (s, 2×CH₃, 6H),3.56 (m, aliphatic protons, 2H), 3.48 (t, J=6.3 Hz, aliphatic protons,2H), 3.08 (t, J=5 Hz, aliphatic protons, 2H), 2.89 (t, J=7 Hz, aliphaticprotons, 2H), 1.95 (m, aliphatic protons, 2H), 1.86 (m, aliphaticprotons, 2H).

[0153] Methyl3-[4-({3-[bis(4-methoxyphenyl)phenylmethoxy]propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}amino)butanoyl]pyrrolo[4,5-e]indoline-7-carboxylate (23).

[0154] To a solution of 22 (3.0 g, 3.37 mmol) in 15 mL anhydrous DMF isadded triethylamine (0.75 mL) followed by methylpyrrolo[4,5-e]indoline-7-carboxylate (Boger, D. L., Coleman, R. S.,Invergo, B. J. (1987) J. Org. Chem. 52, 1521.) (0.8 g, 3.7 mmol). Theresultant solution is stored at room temperature for 20 h. The reactionis analyzed by HPLC to confirm its completeness. DMF is removed on arotary evaporator equipped with an oil pump. The residue, dark syrup issuspended in 50% ethylacetate/hexanes (˜25 mL). The mixture is sonicatedto initiate the crystallization. The crystals are stirred for 15 min,collected by filtration on a sintered glass funnel, washed with methanol(2×30 mL) and dried under vacuum. The yield of the desired product is2.7 g (87%) as a deep-purple solid. ¹H NMR (DMSO-d6) δ 11.93 (d, J=1.7Hz, indole NH, 1H), 8.43 (d, J=2.5 Hz, aromatic proton, 1H), 8.3-8.2 (m,aromatic protons, 2H), 7.85-7.75 (m, aromatic protons, 3H), 7.45-7.18(m, aromatic protons, 10 H), 7.05 (d, J=1.8 Hz, aromatic proton, 1H),6.97 (d, J=9 Hz, aromatic protons, 2H), 6.87 (d, J=9 Hz, aromaticprotons, 4H), 4.12 (t, J=8 Hz, aliphatic protons, 2H), 3.87 (s, esterCH₃, 3H), 3.71 (s, CH₃, 6H), 3.60 (br t, aliphatic protons, 2H), 3.45(br t, aliphatic protons, 2H), 3.29 (br t, aliphatic protons, 2H), 3.08(t, J=5 Hz, aliphatic protons, 2H), 2.5 (br t, obscured by DMSO signal,aliphatic protons, 2H), 1.88 (br m, aliphatic protons, 4H).

[0155] 2,3,4,5,6-pentafluorophenyl3-[4-({3-[bis(4-methoxyphenyl)phenylmethoxy]-propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}amino)butanoyl]-pyrrolo[4,5-e]indoline-7-carboxylate(24)

[0156] 1. Hydrolysis of the methyl ester

[0157] To a solution of 23 (2.67 g, 2.9 mmol) in 25 mL THF are addedmethanol (25 mL) and 5% LiOH, monohydrate in H₂O (10 mL). The resultantsuspension is stirred at 50° C. (bath temperature) for 90 min. by whichtime a clear solution is obtained. TLC analysis shows no startingmaterial. Solvent is removed under vacuum and the product is partitionedbetween CH₂Cl₂ and cold 10% citric acid. The organic phase isneutralized with triethylamine, dried over Na₂SO₄ and concentrated. Theresultant product (amorphous solid) is dried in high vacuum for at least3 h and used in the next step without additional purification.

[0158] 2. PFP ester preparation

[0159] The product obtained in the previous step is dissolved in 100 mLanhydrous DMF. Triethylamine (2 mL) is added followed by PFP-TFA (2 mL,4.4 mmol). The reaction is stirred for 30 min and analyzed by HPLC. Nostarting material, free acid should be observed. DMF is evaporated andthe residue, deep purple syrup is suspended in 100 mL MeOH. Afterstirring for 30 min, a dark precipitate is formed which is collected byfiltration on a sintered glass funnel, washed with methanol (3×20 mL)and dried under vacuum (15-30 h). This procedure yields 2.7 g (94%) ofthe desired product as a purple solid. ¹H NMR (DMSO-d6) δ12.45 (d, J=1.8Hz, indole NH, 1H), 8.43 (d, J=2.5 Hz, aromatic proton, 1H), 8.38 (d,J=9 Hz, aromatic proton, 1H), 8.24 (dd, J₁=9 Hz, J₂=2.5 Hz, aromaticproton, 1H), 7.85-7.75 (m, aromatic protons, 3H), 7.52-7.18 (m, aromaticprotons, 11 H), 6.97 (d, J=9 Hz, aromatic protons, 2H), 6.88 (d, J=9 Hz,aromatic protons, 4H), 4.16 (t, J=8.5 Hz, aliphatic protons, 2H), 3.71(s, CH₃, 6H), 3.61 (br t, aliphatic protons, 2H), 3.47 (br t, aliphaticprotons, 2H), 3.32 (br t, aliphatic protons, 2H), 3.08 (t, J=5 Hz,aliphatic protons, 2H), 2.5 (br t, obscured by DMSO signal, aliphaticprotons, 2H), 1.88 (br mi, aliphatic protons, 4H).

Example 5 2,3,4,5,6-Pentafluorophenyl3-{[3-({3-[4-({3-[bis(4-methoxyphenyl)-phenylmethoxy]propyl}{4-[(2-chloro4-nitrophenyl)diazenyl]phenyl}-amino)butanoyl]pyrrolo[4,-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indolin-7-yl}carbonyl}pyrrolo[4,5-e]indoline-7-carboxylate(25a). where R₁=2−Cl and t=v=3 (Reaction Scheme 6)

[0160] 2-(4-Nitrophenyl)ethyl3-{[3-({3-[4-({3-[bis(4-methoxyphenyl)phenylmethoxy]propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}amino)butanoyl]pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4, 5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indoline-7-carboxylate (25).

[0161] Into a 100 mL round bottom flask is weighed out 1.31 g (1.22mmol) of 24. This is dissolved in 25 mL of dimethylformamide. Then 0.81mL of triethylamine is added, and finally 0.623 g (1.162 mmol) of 17.The reaction mixture is left overnight, then the solution isconcentrated to ˜10 mL and the resultant precipitate is filtered off,using a sintered glass filter funnel. The solid is washed with generousvolumes of methanol (stirring the sludge in the filter with the methanolbefore applying the vacuum) several times and ether. When the effluentis clear and essentially colorless, the deep violet precipitate is driedin vacuo to afford 1.5 g (90%) of the desired product. ¹H NMR (DMSO-d6)δ 11.96 (s, indole NH, 1H), 11.76 (s, indole NH, 1H), 11.69 (s, indoleNH, 1H), 8.43 (d, J-2.4 Hz, aromatic proton, 1H), 8.35-8.20 (m, aromaticprotons, 4H), 8.19 (d, J=9 Hz, aromatic protons, 2H), 7.85-7.75 (m,aromatic protons, 3H), 7.66 (d, J=9Hz, aromatic protons, 2H), 7.45-7.18(m, aromatic protons, 12H), 7.10 (s, aromatic proton, 1H), 7.01 (s,aromatic proton, 1H), 6.99 (m, aromatic protons, 3H), 6.88 (d, J=9 Hz,aromatic protons, 4H), 4.61 (m, aliphatic protons, 6H), 4.14 (t, J=8.5Hz, aliphatic protons, 2H), 3.71 (s, 2×CH₃O, 6H), 3.59 (m, aliphaticprotons, 2H), 3.43 (m, aliphatic protons, 6H), 3.34 (m, obscured bywater signal, aliphatic protons, 2H), 3.22 (m, aliphatic protons, 2H),3.08 (t, J=5 Hz, aliphatic protons, 2H), 2.5 (t, obscured by DMSOsignal, COCH₂−, 2H), 1.89 (br m, aliphatic protons, 4H). Analysis:Calculated: C, 68.27%; H, 4.95%; N, 10.81%. Found: C, 68.08%; H, 4.98%;N, 10.63%.

[0162] 2,3,4,5,6-Pentafluorophenyl3-{[3-({3-[4-({3-[bis(4-methoxyphenyl)phenylmethoxy]propyl}{4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}amino)butanoyl]pyrrolo[4,5-e]indolin-7-yl}carbonyl)pyrrolo[4,5-e]indolin-7-yl]carbonyl}pyrrolo[4,5-e]indoline-7-carboxylate(25a).

[0163] Into a flask is placed 1.0 g (0.73 mmol) of the product from theprevious step, 40 mL of THF, and 2.46 g of DBU. The mixture is stirredat 50 degrees for 4 hours, then removed from the heat and evaporated to15 to 20 ml. About 40 mL of methanol is added to the product and themixture is agitated and sonicated. Then the precipitate is filtered offwith a sintered glass funnel and washed with 40-60 mL of additionalmethanol, followed by a similar amount of ethyl ether, each timestirring the material in the filter prior to applying the vacuum so thatthe effluent soon becomes clear. The product is dried in vacuo for anhour or two before it is used in the next step. The material isdissolved in 20 mL of DMF in a 100 mL flask and stirred to dissolve.Then 0.6 mL (4.3 mmol) of triethylamine is added, followed by 0.6 mL ofPFP-TFA. The reaction mixture is stirred under argon overnight, and thenevaporated to a gum and a ˜10 mL of DMF is added, followed by ˜80 mL ofmethanol. This mixture is swirled and sonicated, and then the product,which precipitates out, is filtered off and dried in vacuo. Yield is85-90%. ¹H NMR (DMSO-d6) δ 12.01 (s, indole NH, 1H), 11.76 (s, indoleNH, 1H), 11.69 (s, indole NH, 1H), 8.43 (d, J-2.4 Hz, aromatic proton,1H), 8.40 (br s, aromatic proton, 1H), 8.35-8.20 (m, aromatic protons,3H), 7.85-7.75 (m, aromatic protons, 3H), 7.59 (d, J=1.2 Hz, aromaticproton, 1H), 7.45-7.18 (m, aromatic protons, 12H), 7.13 (s, aromaticproton, 1H), 6.99 (m, aromatic protons, 3H), 6.88 (d, J=9 Hz, aromaticprotons, 4H), 4.66 (m, aliphatic protons, 4H), 4.14 (t, J=8.5 Hz,aliphatic protons, 2H), 3.71 (s, 2×CH₃O, 6H), 3.59 (m, aliphaticprotons, 2H), 3.43 (m, aliphatic protons, 6H), 3.34 (m, obscured bywater signal, aliphatic protons, 2H), 3.08 (t, J=5 Hz, aliphaticprotons, 2H), 2.5 (t, obscured by DMSO signal, COCH₂—, 2H), 1.89 (br m,aliphatic protons, 4H). Analysis: Found: C, 63.58%; H, 4.13%; N, 9.53%.Calculated, for 2.3 moles of water: C, 63.97%; H, 4.21%; N, 9.44%.

Example 6 DMTrO-Red 13-amide-CDPI₃-CPG (29) (Reaction Scheme 7)

[0164] 3-[(4-Methoxyphenyl)diphenylamino]propan-1-ol (26).

[0165] 4 g (53 mmol) of 3-aminopropanol was dissolved by stirring in 50mL of methylene chloride in an oven dried 250 mL round bottom flask.This solution was stoppered and set aside. 7.7 g (24.9 mmol) ofmonomethoxytrityl chloride (MMT-Cl, Aldrich reagent grade) was dissolvedin another 50 ml of methylene chloride. An oven dried dropping funnelwas fitted to the flask and the MMT-Cl solution was added to the funnel.The MMT-Cl solution was then added to the solution in the flask over ˜10min (some heat develops). After an hour the reaction was analyzed by TLC(1:1 v/v hexanes/ethyl acetate, R_(f) 0.4) and found to be complete.Visualization of TLC spots by ninhydrin spray/heat showed a trace of(faster moving) bis-MMT side product. The reaction mixture was added to200 mL of water standing over 200 mL of methylene chloride in aseparatory funnel. The mixture was shaken and separated into layers; theaqueous layer was discarded and the organic layer was washed with anadditional 200 mL of water. The organic layer was dried over 10-20 g ofsodium sulfate and evaporated to give ˜7 g of the tritylated amine as apale yellow syrup. This compound did not require further purificationand was dried overnight. After several days the syrup solidified. Theproduct was recrystallized from ether-hexanes to give 4.6 g (53% yield)of 26 as a white solid (mp=89.5-90.5 EC). Anal. calcd for C₂₃H₂₅NO₂: C,79.51; H, 7.25; N, 4.03. Found: C, 79.48; H, 7.18; N, 3.98.

[0166]2-[({3-[(4-Methoxyphenyl)diphenylamino]propyl}oxycarbonyl)methoxy]-aceticacid, triethylammonium salt (27).

[0167] 2.72 g (7.83 mmol) of the alcohol (26) was dissolved in 20 mL ofmethylene chloride with 1.3 mL (9.4 mmol) of triethylamine and 1.1 g(9.5 mmol) of glycolic anhydride. The mixture was stirred for 2 h(became homogeneous). TLC showed clean reaction (Rf=0.35 in9:1/methylene chloride:methanol). The solvents were removed byevaporation and the residue was chromatographed on a 1.5×18 inch silicagel column packed with 93% methylene chloride, 5% methanol, and 2%triethylamine. The fractions containing product were combined andsolvent was removed by evaporation. Co-evaporation with dry DMF ensuredremoval of traces of water and of residual volatile solvents. Yield ofthe colorless syrup (27) was assumed to be 100%. The syrup was dissolvedin dry DMF to give a final volume of 23.4 mL (˜0.33 M solution).

[0168] Synthesis of N-MMT diglycolate CPG (28).

[0169] 10 g of LCAA-CPG was combined with 5 mL of a 0.33 M solution of27 in DMF (1.66 mmol) in a 100 mL round bottom flask. A solution of 2.5mL of diisopropylethylamine, 0.11 g (0.8 mmol) of HOBT and 0.63 g (1.66mmol) of HBTU was prepared and added to the CPG. The mixture wasstoppered and swirled for 16 h on an orbital shaker (150 rpm). The CPGwas filtered on a medium porosity sintered glass funnel and washed with2×100 mL of DMF, 2×100 mL of acetonitrile, and 2×100 mL of ether. Tracesof ether were removed in vacuo (oil pump). Unreacted amino groups wereacetylated by treating the CPG with 40 mL of dry pyridine and 5 mL ofacetic anhydride. After swirling for 1.5 h, the CPG was filtered andwashed with 2×100 mL of DMF, 2× 100 mL of acetonitrile, and 2×100 mL ofether. Traces of ether were removed in vacuo (oil pump). The CPG wasanalyzed for MMT loading by treating 3-5 mg of CPG in 25 mL of 1:1/70%perchloric acid:methanol. The absorbance of the released MMT cation wasrecorded at 472 nm and loading level was calculated to be 95.7 :mol/g ofCPG using the equation:

MMT loading (:mol/g) =A472×volume (in mL)×14.3÷wt of CPG (mg)

[0170] Synthesis of CPG 29.

[0171] 4 g of N-MMT diglycolate CPG (28) was weighed into a mediumporosity sintered glass funnel. The CPG was detritylated by treatingwith 25 mL of 3% TCA/DCM. After stirring briefly with a spatula, themixture reacted for 5 min before filtering (turned yellow). The processwas repeated 4 times until the filtrate was colorless. The CPG waswashed with 4×40 mL of methylene chloride. The filtrate was discarded toorganic waste, and the CPG was neutralized by treatment with 40 mL of20% triethylamine in acetonitrile. After briefly stirring with aspatula, the mixture was filtered and washed with 2×40 mL ofacetonitrile, and 2×40 mL of ether. Traces of ether were removed invacuo (oil pump). The de-tritylated CPG was used immediately for thefollowing immobilization reaction.

[0172] 0.259 g (180 :mol) of 25a was shaken with 12 mL of dry DMSO in a15 mL polypropylene tube. After 15 min, the dark purple solution wasadded to 4 g of detritylated diglycolate CPG (in a 50 mL round bottomflask). This corresponds to an offering ratio of 45 :mol PFP ester pergram of CPG. An additional 5 mL of DMSO was added to the polypropylenetube to dissolve residual PFP ester and the solution was added to theCPG. 2 mL of triethylamine was added and the mixture was stoppered andswirled on an orbital mixer for 14 h. The CPG was filtered and washedwith 2×50 mL of DMSO, 2×50 mL of acetonitrile, and 2×50 mL of ether.Traces of ether were removed in vacuo (oil pump). Unreacted amino groupswere acetylated by treating the CPG with 10 mL of dry pyridine and 3 mLof acetic anhydride. After swirling for 6 h, the CPG was filtered andwashed with 2×50 mL of DMF, 2×50 mL of acetonitrile, and 2×50 mL ofether. Traces of ether were removed in vacuo (oil pump). The CPG wasanalyzed for DMT loading by treating 3-5 mg of CPG in 25 mL of 1:1/70%perchloric acid:methanol. The absorbance of the released DMT cation wasrecorded at 498 nm and loading level was calculated to be 45 :mol/g ofCPG using the equation:

DMT loading (:mol/g)=A₄₉₈×volume (in mL)×14.3÷wt of CPG (mg)

Example 7 Synthesis of FL-ODN-Red 13-amide-CDPI₃ (30).

[0173]

[0174] The oligonucleotides were synthesized on the CPG 29 usingstandard phosphoramidite coupling chemistry except that the standard 0.1I₂ oxidizing solution was diluted to 0.01-0.015 to avoid iodination ofthe MGB moiety. FAM and TET were incorporated at the 5′end using thecorresponding phosphoramidites available from Glen Research.

Example 84-{[N-6-{[Bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}hexyl)carbamoyl]methyl}-2-oxo-2H-chromen-7-yl2,2-dimethylpropanoate (34a).

[0175] N-(6-Hydroxyhexyl)-2-(7-hydroxy-2-oxo(2H-chromen-4-yl))acetamide(32a).

[0176] (7-Hydroxy-2-oxo-2H-chromen-4-yl)-acetic acid methyl ester (1)was synthesized according to Baker et al. (J. Chem. Soc.; 1950; 170,173.).

[0177] A solution of 31 (2.0 g, 8.5 mmol) and 6-aminohexanol (4.0 g,34.1 mmol) in 15 mL of DMF was heated at 80° C. for 24 h. DMF wasevaporated under vacuum to afford the mixture of the product and theexcess 6-aminohexanol as a viscous syrup. Chromatography on silicaeluting with 10% MeOH/CH₂Cl₂ and evaporation of the pure productfractions afforded a white solid which was washed with ether and driedunder vacuum. The yield was 2.05 g (75%).

[0178] 4-{[N-(6-Hydroxyhexyl)carbamoyl]methyl}-2-oxo-2H-chromen- 7-yl2,2-dimethylpropanoate (33a).

[0179] To a solution of 32a (2.0 g, 6.3 mmol) in 20 mL of dry pyridinewas added 4,4′-dimethoxytriphenylmethyl chloride (3.0 g, 8.9 mmol). Thesolution was kept at room temperature for 1 h, TLC analysis (ethylacetate, R_(f)˜0.7) showed complete reaction (protection of the primaryhydroxy group). To this solution was added trimethylacetic anhydride(2.0 mL, 9.9 mmol) followed by triethylamine (5 mL) and4-(dimethylamino)pyridine (0.3 g). The mixture was stirred for 5 h, TLCanalysis showed complete protection of the phenol group (R_(f)˜0.9,ethyl acetate). Methanol was added to quench excess anhydride. Pyridinewas removed by evaporation under vacuum and co-evaporation with xylene.The product obtained was partitioned between ethyl acetate and 2%NaHCO₃, the organic phase was concentrated under vacuum to give thecrude DMT protected 33a.

[0180] To remove the DMT group the DMT derivative was dissolved in 100mL of 10% MeOH in CH₂Cl₂ and treated with 0.5 mL of trifluoroaceticacid. After being stirred for 1 h, the reaction mixture was neutralizedwith triethylamine (0.7 mL) and concentrated. The resultant viscous oilwas partitioned between ethyl acetate and water. The organic layer wasdried over Na₂SO₄ and concentrated. The solid obtained was suspended inether (50 mL) and stirred for 30 min. The desired product was theinsoluble material, and was collected by filtration, washed with etherand dried. The yield of the title product 33 was 1.6 g (64%).

[0181]4-{[N-(6-{[Bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}hexyl)carbamoyl]methyl}-2-oxo-2H-chromen-7-yl2,2dimethylpropanoate (34).

[0182] To a solution of 33a (0.6 g, 1.5 mmol) in 10 mL of anhydrousCH₂Cl₂ was added triethylamine (0.4 mL) followed by 2-cyanoethyldiisopropylchlorophosphoramidite (0.35 mL, 1.6 mmol). The solution waskept at room temperature for 1 h and treated with 0.1 mL of MeOH. Thesolvent was evaporated and the residue was partitioned between ethylacetate and saturated NaHCO₃. The organic phase was washed withsaturated NaCl, dried over Na₂SO₄ and concentrated. The crude productwas chromatographed on silica eluting with 5% triethylamine in ethylacetate. Concentration of the pure product fractions and drying undervacuum afforded 0.59 g (65%) of 34 as a colorless, viscous oil.

Example 98-(3-{[bis(methylethyl)amino[(2-cyanoethoxy)phosphinooxy}propyl)-7-oxophenoxazin-3-yl2,2-dimethylpropanoate (37a). (Reaction Scheme 9)

[0183] 7-Hydroxy-2-(3-hydroxypropyl)phenoxazin-3-one (35a).

[0184] A suspension of 4-nitrosorecorcinol (4.5 g, 32.4 mmol),4-(3-hydroxypropyl)benzene-1,3-diol (Forchiassin, M.; Russo, C., J.Heterocyc. Chem. 20, 1983, 493-494.) (4.0 g, 23.8 mmol) and MnO2 (2.5 g,17.6 mmol) in 50 mL of MeOH was cooled to ˜0° C. (ice bath). To thissuspension was added dropwise 2.5 mL of conc. H₂SO₄ and the reaction wasstirred at room temperature for 5 h. The precipitated red resazurincompound was collected by filtration, washed with methanol and dried.The yield was 5.5 g. This product was not homogeneous, it wascontaminated with resorufin compound and manganese salts.

[0185] The crude resazurin compound was suspended in a mixture of 200 mLof water and 50 mL of conc. NH₄OH. Zinc dust (2.0 g) was added and thesuspension was stirred for 20 min. The resultant purple mixture wasfiltered, the filtrate was vigorously stirred on air to oxidize theleuco resorufin, the product of partial over reduction. The reaction wasacidified with acetic acid, the brown solid formed was collected byfiltration washed with water and dried. The yield was 2.1 g. Thematerial contained ˜50% of2,3,4-trihydro-2H-pyrano[3,2-b]phenoxazin-9-one, product ofintramolecular cyclization which had been carried over from the firststep. The rest of the material was the desired title compound 35.

[0186] 8-(3-hydroxypropyl)-7-oxophenoxazin-3-yl 2,2-dimethylpropanoate(36b).

[0187] A suspension of 35a (2.0 g) in 50 mL of pyridine was treated with4,4′-dimethoxytriphenylmethyl chloride (5.0 g, 14.8 mmol) and stirredfor 5 h. The mixture was filtered to remove some insoluble material andthe filtrate was treated with trimethylacetic anhydride (2 mL). Thesolution was stirred for 15 h and MeOH (2 mL) was added to quench excessanhydride. After being stirred for 3 h, the reaction mixture wasconcentrated under vacuum. Residual pyridine was removed byco-evaporation with triethylamine and xylene. The resultant crudeproduct 36a was chromatographed on silica eluting with 50% ethylacetate/hexane.

[0188] The DMT derivative was dissolved in 100 mL of 10% MeOH/CH₂Cl2 andtreated with 0.5 mL of trifluoroacetic acid. After 1 h, triethylamine (2mL) was added and the solution was concentrated. Chromatography onsilica (ethyl acetate) and drying afforded 0.38 g of the desired productas an orange solid.

[0189] 8-(3-{[bis(methylethyl)amino](2-cyanoethoxy)phosphinooxy}propyl)-7-oxophenoxazin-3-yl 2,2-dimethylpropanoate (37a).

[0190] 36b (0.38 g, 1.1 mmol) was dissolved in 6 mL of anhydrous CH₂Cl₂.Triethylamine (1.5 mL) was added followed by 2-cyanoethyldiisopropylchlorophosphoramidite (0.29 mL, 1.3 mmol). The solution waskept at room temperature for 30 min, MeOH (0.1 mL) was added and thereaction was concentrated under vacuum. The residue obtained waspartitioned between ethyl acetate and NaHCO₃. The organic phase waswashed with saturated NaCl, dried over Na₂SO₄ and concentrated to affordthe crude amidite. It was dissolved in 2 mL of ether and added dropwiseto ˜50 mL of hexane. The resultant orange solid was collected byfiltration, washed with hexane and dried. The yield was 0.4 g.

Example 103-{[(tert-Butyl)(methylethyl)amino][2-(4-{3-butyl-7-[(4-methyphenyl)-carbonyl]-2,4,6,8-tetraoxo-1-(phenylcarbonyl)(1,3,5,7,9,10-hexahydro-pyrimidino[5′,4′-5,6]pyridino[2,3-d]pyrimidin-10-yl)}phenyl)ethoxy]-phosphinooxy}propanenitrile(PPT) 44 (Reaction Scheme 10)

[0191] 3-n-Butyl-6-[4-(2-hydroxyethyl)aminophenyl]uracil 40.

[0192] A mixture of 6-chloro-3-n-butyluracil (10.4 g, 51.3 mmol),2-(4-aminophenyl)ethanol (10.0 g, 72.9 mmol) and ethyldiisopropylamine(18 ml, 0.1 mol) was heated with stirring under argon on a 150° oil bathfor 1 hr 20 min. The mixture was cooled to room temperature, dilutedwith 50 ml of water, treated with 10 ml of acetic acid and stirred forcrystallization overnight. A precipitated solid was filtered, washedwith 2% acetic acid, dried on filter and dissolved in 100 ml of hot 96%ethanol. To the solution 100 ml of hot water was added followed by 1.0 gof charcoal. The mixture was filtered hot and crystallized on ice.Yellow solid was collected by filtration and dried in vacuum to yield10.7 g of 40, mp 207-208° C. ¹H NMR (DMSO-d₆) δ0.88 (t, 3H, J=7.3 Hz,CH₃), 1.25 (m, 2H, CH₂), 1.46 (m, 2H, CH₂), 2.70 (t, 2H, J=6.8 Hz, CH₂),3.60 (dd, 2H, J=11.8, 6.8 Hz, CH₂), 3.68 (t, 2H, J=7.3 Hz, CH₂), 4.62(t, 1H, J=5.3 Hz, OH), 4.73 (d, 1H, J=1.8 Hz, 5-H), 7.10 (d, 2H, J=8.4Hz, ArH), 7.23 (d, 2H, J=8.4 Hz, ArH), 7.10 (s, 1H, NH), 10.37 (s, 1H,NH).

[0193] 3-n-Butyl-10-[(2-hydroxyethyl)phenyl]pyrido[2,3-d;6,5-d′]dipyrimidine-2,4,6,8-(3H, 7H, 9H, 10H)-tetrone.41

[0194] A solution of 40 (6.6 g, 20 mmol) and5-formyl-2,4,6-trichloropyrimidine (5.85 g, 27.7 mmol) in 80 ml of dryDMF was stirred at RT for 8 hr and slowly diluted with 80 ml of water.The solution produced a solid upon refrigeration for 2 days. The productwas isolated by filtration, washed with cold 50% ethanol (50 ml) and 25%ethanol (50 ml) and dried in vacuum to yield 8.16 g (96%) 41 as acolorless solid, mp 205-215 ° C. (decomp). ¹H NMR (DMSO-d₆) δ 0.88 (t,3H, J=7.2 Hz, CH₃), 1.27 (m, 2H, CH₂), 1.48 (m, 2H, CH₂), 281-2.90 (m,2H, CH₂), 3.71-3.85 (m, 4H, CH₂), 4.50 (br. s, 5H, OH, NH, H₂O), 7.26(d, 2H, J=8.4 Hz, ArH), 7.44 (d, 2H, J=8.4 Hz, ArH), 8.62 (s, 1H, 5-H).

[0195] 3-n-Butyl-5,10-dihydro-10-[(2-hydroxyethyl)phenyl]pyrido[2,3-d,-6,5-d′]dipyrimidine-2,4,6,8-(1H,3H, 7H,9H, 10H)-tetrone. 42

[0196] To a suspension 41 (7.91 g, 18.7 mmol) in 300 ml of 25% aq. NH₃was added Na₂S₂O₄ (13.8 g, 85%, 67 mmol) and slowly heated to 60° withstirring. The mixture was stirred at 60° for 40 min, diluted with water(100 ml) and stirred for additional 1 hr at the same temperature. Aclear solution formed. The solution was partially evaporated to one halfof its original volume, cooled with ice and neutralized with 50 ml ofacetic acid to pH 5 to form a precipitate. The mixture was kept inrefrigerator for complete crystallization, filtered and washed with coldwater. The solid was dried in vacuum to yield 7.32 g (92%) of 42 as awhite solid, mp 182-210 ° C. (decomp). ¹H NMR (DMSO-d6) δ0.86 (t, 3H,J=7.3 Hz, CH₃), 1.23 (m, 2H, CH₂), 1.42 (m, 2H, CH₂), 2.80 (t, 2H, J=6.6Hz, CH₂), 3.14 (s, 2H, 5-CH₂), 3.68 (m, 4H, ArCH₂CH₂), 4.64 (t, 1H, OH),7.25 (d, 2H, J=8.3 Hz, ArH), 7.33 (d, 2H, J=8.3 Hz, ArH), 7.73 (br. s,3H, NH).

[0197] Solid 42 (1.2 g, 2.82 mmol) was evaporated with pyridine (10 ml),suspended in pyridine (13 ml), treated with Me₃SiCl (2.2 ml, 17.3 mmol)and stirred under argon at ambient temperature for 30 min. The reactionmixture was cooled with ice and treated slowly with toluoyl chloride (5ml, 28.8 mmol). Stirring was continued at room temperature for 2 hr, andthe solvent evaporated. The residue was treated with acetic acid (10 ml)followed by addition of water (10 ml). Precipitated oil was extractedwith hexanes (3×50 ml), and the residue that was insoluble in hexaneswas evaporated with water. The residue was suspended in 96% ethanol (10ml) and filtered to recover 0.3 g of the starting material. The motherliquor was diluted with water to precipitate bis-toluoyl derivative asan oil. The oil was dried in vacuum to give 0.96 g (52%) of 43 as asolid foam. This compound without further purification was convertedinto phosphoramidite by the following procedure. The solid wasevaporated with acetonitrile, dissolved in 25 ml of dichloromethane,treated with diisopropylammonium tetrazolide (0.54 g, 3.13 mmol)followed by 2-cyanoethyl tetraisopropylphosphorodiamidite (0.88 g, 2.9mmol). The reaction mixture was stirred under argon for 1 hr, treatedwith methanol (1 ml), taken into EtOAc (100 ml), washed with sat. NaClsolution and dried over Na₂SO₄. The solution was evaporated, purified byHPLC on silica gel column using a gradient system 0-50% B;CH₂Cl₂-hexanes-NEt₃ (15:30:1) (A); EtOAc (B); detected at 320 nm. Themain fraction was evaporated giving a colorless foam, 0.79 g (33%) ofAG1 phosphoramidite 44. ¹H NMR (CDCl₃) δ0.92 (t, 3H, J=7.3 Hz, CH₃),1.07-1.42 (m, 14H, 4×CH₃ (i-Pr), CH₂ (Bu)), 1.50-1.65 (m, 2H, CH₂ (Bu)),2.35-2.60 (m, 2H, CH₂CN), 2.40 (s, 3H, CH₃Ar), 2.46 (s, 3H, CH₃Ar),2.95-3.13 (m, 4H, 2×CH (i-Pr), CH₂ (Bu)), 3.45-3.60 (m, 2H, OCH₂),3.80-4.02 (m, 4H, ArCH₂CH₂), 3.82 (s, 2H, 5-CH₂), 7.15-7.35 (m, 8H, ArH(Tol)), 7.45 (s, 1H, NH), 7.73 (br. s, 3H, NH), 7.95 (d, 2H, J=8.0 Hz,ArH), 8.05 (d, 2H, J=8.0 Hz, ArH). ³¹p NMR (CDCl₃) δ (ppm, H₃PO₄) 143.2(s).

Example 11N-{3-[4-[(1Z)-1-aza-2-dimethylamino)prop-1-enyl]-1-(5-{]bis(4-methoxyphenyl)phenylmethoxy}methyl}-4-{[bis(methylethyl)amino](2-cyanoethyl)phophinooxy}oxolan-2-yl)pyrazolo[5,4-d]pyrimidin-3-yl]propyl}[2-({4-[(2-chloro-4-nitrophenyl)diazenyl]phenyl}ethylamino)-ethoxy]carboxamide(50) (Reaction Scheme 11)

[0198]4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-(3-trifluoroacetimido-propyn-1-yl)pyrazolo[3,4-d]pyrimidine (46; n=1).

[0199] To a mixture of 45 (1.96 g, 5.20 mmol), CuI (103 mg, 0.54 mmol)and tetrakis[triphenylphosphine]palladium[0] (317 mg, 0.276 mmol) in 10ml of anhydrous DMF was added anhydrous triethylamine (1.1 ml) followedby propargyl trifluoroacetimide (1.50 g, 9.88 mmol). The reactionmixture was stirred under argon for 4 h. The solvent DMF was removed byevaporation and the residual oil was purified by silica gelchromatography eluting with 7% methanol in ethyl acetate. The productfractions were pooled and evaporated affording a foam: 2.16 g (99%)yield.

[0200]4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-3-(3-aminopropyl)pyrazolo[3,4-d]pyrimidine(47; n=1).

[0201] To a solution of 46 (2.10 g, 5.25 mmol) in 50 ml of ethanol,containing 0.300 mg of 5% palladium on carbon (preactivated with formicacid), was added 1.0 ml of 4 M triethylammonium formate buffer (pH 6.5).The mixture was shaken under 40 psi of hydrogen gas for 18 h. Themixture was filtered through Celite and the filtrate was evaporatedaffording a solid. 1.8 g (85%) yield.

[0202] The solid was stirred in 15 ml of concentrated ammonium hydroxide(sealed flask) for 12 h and then evaporated to dryness. The solid (47)was evaporated from dry acetonitrile and stored under vacuum: 1.74 gyield.

[0203] Synthesis of 48 (n=1, q=2, R₅=CH₃CH₂—, R₅, R₅=H, R₁=2-Cl,R₅=4-NO₂).

[0204] A solution of 47 (0.90 g, 2.92 mmol) and 7 (1.59 g, 2.92 mmol)was stirred in 5.0 ml of anhydrous dimethylformamide, containing 1.0 mlof triethylamine, at 50° C. for 1.0 h. The solution was evaporated todryness and the residue was purified by silica gel chromatographyeluting with a gradient of 0-20% methanol in ethyl acetate. The productfractions were evaporated affording an amorphous solid: 0.74 g (37%)yield.

[0205] Synthesis of 49 (n=1, q=2, R₅=CH₃CH₂—, R₅, R₅=H, R₁=2-Cl,R₅=4—NO₂).

[0206] To a solution of 48 (0.71 g, 1.03 mmol) and N,N-dimethylacetamidedimethylacetal (1.9 ml) in 5.0 ml of dimethylacetamide was added 2.0 mlof triethylamine. The solution was stirred for 18 hrs and thenevaporated to dryness affording an oil: 0.75 g (100%) yield.

[0207] Synthesis of 50 (n=1, q=2, R₅=CH₃CH₂—, R₅, R₅-H, R₁=2-Cl,R₅=4-NO₂).

[0208] Dimethoxytrityl chloride (0.42 g) was added to a solution of 49(0.75 g, 1.03 mmol) in 10 ml of dry pyridine. The solution was stirredfor 4.0 hr under argon and then poured into 200 ml of 5% sodiumbicarbonate solution. The product was extracted with 300 ml of ethylacetate. The extract was dried over sodium sulfate and evaporated. Theresidue was purified by silica gel chromatography eluting with 10%methanol in ethyl acetate (1% triethylamine). The product fractions wereevaporated affording a foam: 556 mg (57%) yield.

[0209] To a solution of the 5′-dimethoxytrityl derivative (540 mg, 0.567mmol) in 15 ml of anhydrous methylene chloride, containing 0.30 ml ofdiisopropylethylamine, was added 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.25 ml). After stirring for 30minutes under argon at 25° C. the solution was treated with 1.0 ml ofmethanol and diluted with 200 ml of ethyl acetate. The solution waswashed with 200 ml of 5% sodium bicarbonate solution and dried oversodium sulfate and evaporated. The crude product was purified by silicagel chromatography eluting with 5% methanol in ethyl acetate (2%triethylamine). The product fractions were evaporated affording a foam:453-mg (76%) yield.

Example 12 Synthesis of Fluorogenic Oligodeoxynucleotide Probes.

[0210] The 3′- DPI₃ probes were prepared by automated DNA synthesis froma DPI₃-modified glass support using methods described earlier (Lukhtanovet al. Biorg. Chem., 7: 564-567 (1996)). Oligonucleotide synthesis wasperformed on an ABI 394 synthesizer according to the protocol suppliedby the manufacturer except that 0.015 M (instead of the standard 0.1 M)iodine solution was utilized in the oxidation step to avoid iodinationof the CDPI₃ moiety. To prevent extension during PCR, probes without 3′-CDPI₃ were prepared with the 3′-hydroxyhexyl phosphate as previouslydescribed (Gamper et al. Biochem. 36: 14816-14826 (1997)). The quencherphosphoramidites were added to the CPG and standardβ-cyanoethylphosphoramidites and reagents (Glen Research, Sterling, Va.)were used in oligonucleotide synthesis. 6-Carboxyfluorescein (6-FAM)phosphoramidite (Glen Research) was used to introduce the 5′-reporterdyes. Alternatively TAMRA-dU phosphoramidite (Glen Research), cy3 or cy5phosphoramidite (Glen Research), resorufin phosphoramidite, coumarinphosphoramidite, or AG phosphoramidite was used to introduce theindicated 5′-fluorophore. 5′-Hexylamine phosphoramidite (Glen Research)was incorporated into certain ODNs for post-synthetic conjugation of the3′-quencher dye tetramethylrhodamine (TAMRA). After deprotection, alloligonucleotides were reverse-phase HPLC purified and isolated as thesodium salts by butanol concentration/sodium perchlorate precipitation(Milesi et al. Methods Enzym. 313: 164-173 (1999)).

Example 13 Post-synthetic Conjugation of ODNs with TAMRA.

[0211] TAMRA NHS ester (Glen Research) was used to acylate thehexylamine linkers in certain ODNs according to the protocol supplied bythe manufacturer. The resulting CDPI₃-probes with two conjugated dyeswere purified by denaturing gel electrophoresis using 8% polyacrylamide.The desired bands were excised and the gel slices were incubatedovernight at 37° C. in 10 mL of 100 mM Tris-HCl, 10 mM triethylammoniumchloride, 1 mM EDTA (pH 7.8). The products were isolated from theextract by reverse phase HPLC, butanol concentration and sodiumperchlorate precipitation. The pellets were dissolved in water and theconcentrations were determined spectrophotometrically. A nearestneighbor model (Cantor, et al. Biopolymers 9: 1059-1077 (1970) wasapplied to calculate extinction coefficients (ε₂₆₀) of ODNs. For theconjugates and probes, extinction coefficients were calculated as a sumof ε260 for the ODN and the incorporated residues of DPI₃ (68,000 M⁻¹,cm⁻), 6-FAM (22,800 M⁻, cm⁻), TAMRA (34,000 M⁻, cm⁻¹) and quencher(11,300 M⁻, cm⁻¹).

Example 14 Digestion of Oligonucleotides by Snake VenomPhosphodiestaerase.

[0212] Oligonucleotides were digested with snake venom phosphodiesterase(PDE) to study the fluorescence quenching potential of variousquenchers. 200 nM of oligonucleotide was taken in a buffer containing 40mM of NaCl, 20 mM of Tris (pH 8.9), 5mM of MgCl₂ and 0.025% of BSA.Initial fluorescence was read on a LS50B fluorimeter (Perkin-ElmerCorporation, Foster City, Calif.) before the addition ofphosphodiesterase (Pharmacia, Piscataway, N.J.) 54 units of enzyme wasadded to the reaction mixture and incubated at 37 ° C. for 16 hrs. Thefinal fluorescence was then measured using the LS50B. The ratio of finalfluorescence to the initial fluorescence represents the signal to noiseratio (S/N) of the quenchers. Independently the kinetics of digestionreactions were monitored using the LS50B to determine the time requiredfor complete digestion of oligonucleotides.

Example 15 5′Nuclease PCR Assay.

[0213] CDPI₃-conjugated oligonucleotides were conjugated with afluorophore, FAM at the 5′ end and various quenchers were conjugatedthrough a linker at the 3′ end by the methods discussed above. 5′nuclease assays were performed with the above oligonucleotides todetermine the quenching ability of the various quenchers underinvestigation. Fluorescent monitoring was performed in an IdahoTechnologies LC-24 LightCycler. Each reaction contained PCR buffer (40mM NaCl, 20 mM Tris HCl, pH 8.9, 5 mM MgSO₄, 0.05% bovine serumalbumin), 125 mM each dNTP, 0.5 mM each primer, 0.1 mM fluorescent CDPI₃probe, 0.5 U/10 mL Taq polymerase and 0.1 ng/10 mL of synthetic DNA astemplate. The cycling program was 50 cycles (or as indicated) of 2 secat 95EC, then 30 sec at the extension temperature (55-70E).

1 5 1 15 DNA Artificial Sequence ODN sequence 1 gagggatgta aaaat 15 2 13DNA Artificial Sequence ODN sequence 2 gtcctgattt tac 13 3 26 DNAArtificial Sequence PCR primer 1 3 gtactttcaa ttcatggagc atacct 26 4 21DNA Artificial Sequence PCR primer 2 4 atggccttgt accgatgctg a 21 5 14DNA Artificial Sequence Non-cleavable MGB-probe 5 atatctagcg ttga 14

What is claimed is:
 1. An oligonucleotide conjugate having the formulaFL-ODN-Q where ODN is an oligonucleotide or nucleic acid; FL is afluorophore moiety covalently attached to the ODN through a linkerhaving the length of 0 to approximately 30 atoms, and Q is a quenchermoiety covalently attached to the ODN through a linker having the lengthof 0 to approximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′),CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n′)CH₃where n″=0 to 5, and where the quencher moiety is attached to the linkerthrough the valence bond designated a.
 2. An oligonucleotide conjugatein accordance with claim 1 where R₀ is H, R₁ is NO₂ in the 4 position ofthe benzene nucleus, R₂ is H or Cl in the 2 position of the benzenenucleus, and R₃ and R₄ are hydrogen and R₅ is ethyl.
 3. Anoligonucleotide conjugate in accordance with claim 1 where the quenchermoiety and the linker attaching it to the ODN comprises the structuresselected from the moieties shown by the formulas Q-1, Q-2 and Q-3

where q is 1 to 20, X is —O—, —OCH₂— or —CH₂—; t and v independently are1 to 20, r and s independently are 1 to 20, and the conjugated quencherand linker moiety is attached to the ODN through one of the valencebonds designated a or b
 4. An oligonucleotide conjugate in accordancewith claim 3 further comprising a minor groove binder moiety attached tothe quencher-linker conjugate through one of the valence bondsdesignated a or b.
 5. An oligonucleotide conjugate in accordance withclaim 1 where the quencher moiety and of the linker attaching it to theODN comprises the structures selected from the moieties shown by theformulas Q-4, and Q-5

where R₆ is —(CH₂)_(n*) where n^(*) is 1 to 20, and t and vindependently are 1 to 20, and where the quencher moiety is attached tothe ODN through the valence bond designated a.
 6. A phosphoramiditereagent for preparing an oligonucleotide-fluorophore-quencher conjugate,the reagent including the moiety

where R₀, R₁, R₂, R₃ and R₄ are independently —H,halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n),CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n′),CH₃where n″=0 to 5, and a bis(methylethyl)amino](2-cyanoethoxy)phosphinooxymoiety covalently linked thereto.
 7. A phosphoramidite reagent inaccordance with claim 6 having the formula selected from the groupconsisting of the formulas designated PA-1, PA-2 and PA-3

where R₀, R₁, R₂, R₃ and R4 are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′),CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n″)CH₃where n″=0 to 5, q is 1 to 20, X is —O— or —CH₂—; t, v, r and sindependently are 1 to 20, and X₂ is H or dimethoxytrityl,methoxytrityl, trityl or an acid labile blocking group.
 8. Aphosphoramidite reagent in accordance with claim 7 that has the formuladesignated PA-1.
 9. A phosphoramidite reagent in accordance with claim 7that has the formula designated PA-2.
 10. A phosphoramidite reagent inaccordance with claim 7 that has the formula designated PA-3.
 11. Aphosphoramidite reagent in accordance with claim 7 where R_(o) is H, R₁is NO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2position of the benzene nucleus, and R₃ and R₄ are hydrogen and R₅ isethyl.
 12. A covalently linked solid support and quencher conjugatesuitable for oligonucleotide synthesis, having the structure

where CPG stands for a polymeric solid support; LINKER is a moietyhaving the length of 1 to approximately 30 atoms and linking thediphenylazo moiety to the CPG; X₂ is OH or, dimethoxytityl,methoxytrityl, trityl or an acid labile blocking group; R₀, R₁, R₂, R₃and R4 are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ wheren=0 to 5, —NO₂, —SO₃, —N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN, andR₅=—H or —(CH₂)_(n″)CH₃ where n″=0 to
 5. 13. A covalently linked solidsupport and quencher conjugate in accordance with claim 12 selected fromthe structures

where R₆ is —(CH₂)_(n*) where n* is 1 to 20, and q, r, t and vindependently are 1 to
 20. 14. A covalently linked solid support andquencher conjugate in accordance with claim 13 where R_(o)is H, R₁ isNO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 positionof the benzene nucleus, and R₅ is ethyl.
 15. An oligonucleotideconjugate having the formula FL-ODN-Q-MGB where ODN is anoligonucleotide or nucleic acid; FL is a fluorophore covalently attachedto the ODN through a linker having the length of 0 to approximately 30atoms, and Q is a quencher moiety covalently attached to the ODN througha linker having the length of 0 to approximately 30 atoms, the quenchermoiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —H,halogen,—O(CH2)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H, —(CH₂)_(n″)CH₃ or—(CH₂)_(n″)— where n″=0 to 5, and MGB is minor groove binder moietycovalently attached to the ODN moiety or to the quencher moiety througha linker having the length of 0 to approximately 30 atoms.
 16. Anoligonucleotide conjugate in accordance with claim 15 where the MGBmoiety is attached to the quencher moiety, and the covalently bonded MGB-Q moiety has the structure

where t and v independently are 1 to 20, and the valence bond designateda attaches the MGB-Q moiety to the ODN moiety.
 17. An oligonucleotideconjugate in accordance with claim 16 where R_(o) is H, R₁ is NO₂ in the4 position of the benzene nucleus, R₂ is H or Cl in the 2 position ofthe benzene nucleus, and R₃ and R₄ are hydrogen.
 18. A covalently bondedminor groove binder and quencher reagent for oligonucleotide synthesis,having the formula

where R₀, R₁, R₂, R₃ and R₄ are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃, —N[(CH₂)_(n′)CH₃]₂where n′=0 to 5 or —CN, and t and v independently are 1 to 20; X₂ is Hor dimethoxytrityl, methoxytrityl, trityl or an acid labile blockinggroup, and X₃ is pentafluorophenyloxy, or NH-LINKER-CPG or O-LINKER-CPGwhere CPG is a polymeric solid support and LINKER is a linking moietyhaving a length of approximately 0 to 30 atoms linking the tricyclicmoiety to the CPG.
 19. A covalently bonded minor groove binder andquencher reagent in accordance with claim 18 wherein X₃ ispentafluorophenyloxy.
 20. A covalently bonded minor groove binder andquencher reagent in accordance with claim 18 wherein X₃ is NH-LINKER-CPGor O-LINKER-CPG.
 21. A covalently bonded minor groove binder andquencher reagent in accordance with claim 18 where R_(o) is H, R₁ is NO₂in the 4 position of the benzene nucleus, R₂ is H or Cl in the 2position of the benzene nucleus, R₃ and R₄ are hydrogen and v=t=3. 22.An oligonucleotide conjugate having the formula FL-ODN-Q where ODN is anoligonucleotide or nucleic acid; Q is a quencher moiety covalentlyattached to the ODN through a linker having the length of 0 toapproximately 30 atoms, and FL is a fluorophore covalently attached tothe ODN through a linker having the length of 0 to approximately 30atoms, said fluorophore moiety having the structure selected from thegroup designated FL-1, FL-2 and FL-3,

wherein R₈and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂, or—CN;—OR_(nn), —SR_(nn), —OR_(nn), —NHR_(nn),—N[R_(nn)]2 where R_(nn) isindependently H, an alkyl group of 1 to 10 carbons or an alkanoyl groupof 1 to 10 carbons; R₁₀, and R₁₁ independently are H, —CN , —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons; R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker.
 23. Anoligonucleotide conjugate in accordance with claim 22 where thefluorophore has the formula designated FL-1.
 24. An oligonucleotideconjugate in accordance with claim 23 where R₈ is OC(O)CH(CH₃)₂ and R₉is H.
 25. An oligonucleotide conjugate in accordance with claim 22 wherethe fluorophore has the formula designated FL-2.
 26. An oligonucleotideconjugate in accordance with claim 25 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁is H.
 27. An oligonucleotide conjugate in accordance with claim 22 wherethe fluorophore has the formula designated FL-3.
 28. An oligonucleotideconjugate in accordance with claim 28 where R₁₅ is methyl and R₁₆ isn-propyl.
 29. An oligonucleotide conjugate in accordance with claim 22where the quencher moiety comprises the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃, —N[(CH₂)_(n′)CH₃]₂where n′=0 to 5 or —CN, and R₅=−H or —(CH₂)_(n″)CH₃ where n″=0 to
 5. 30.An oligonucleotide conjugate in accordance with claim 22 comprising anadditional minor groove binder moiety (MGB) attached to the quenchermoiety through a linker having the length of 0 to approximately 30atoms, whereby the oligonucleotide conjugate has the formulaFL-ODN-Q-MGB.
 31. An oligonucleotide conjugate of the formula

wherein R₀, R₁, R₂, R₃ and R₄ are independently —H,halogen,—O(CH₂)_(n*)CH₃, —(CH₂)_(n*)CH₃ where n*=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN; FL is a fluorophore moietywith emission wavelengths in the range of about 300 to about 800 nm; Kis a linker containing 1 to approximately 30 atoms selected from thegroup consisting of C, O, N, S, P and H; [A-B]_(n) symbolizes an ODN,DNA, RNA or PNA or any combination thereof, where A is the sugarphosphate backbone where the sugar and the phosphate may independentlybe modified; B is a heterocyclic base, where B is independently selectedfrom purine, pyrimidine, pyrazolo[3,4-d]pyrimidine, 7-substitutedpyrazolo[3,4-d]pyrimidine- , 7-deazapurine, 7-substiuted 7-deazapurine,and modified purine- and pyrimidine-bases, and where the DNA, RNA, PNAor ODN can include any combinations of these bases, and and n is thenumber of nucleotide units in said DNA, RNA, PNA or ODN; W is a linkerof a length of 0 to approximately 30 atoms, selected from the groupconsisting of C, O, N, S, P and H, and m is an integer having the valuesof 1 to
 20. 32. An oligonucleotide conjugate in accordance with claim 31where R_(o) is H, R₁ is NO₂ in the 4 position of the benzene nucleus, R₂is H or Cl in the 2 position of the benzene nucleus, and R₃ and R₄ arehydrogen.
 33. An oligonucleotide conjugate in accordance with claim 31where said fluorophore moiety has the structure selected from the groupdesignated FL-1, FL-2 and FL-3,

where R₈ is OH or O-alkanoyl where the alkanoyl group has 1 to 10carbons; R₉ is H or alkyl of 1 to 10 carbons; R₁₀ and R₁₁ independentlyare H, —OR₁₂, —NHR₁₃, halogen,—O(CH₂) _(n)CH₃, —(CH₂)_(n)CH₃, —NO₂,—SO₃, —C(═O)NH₂, —N[(CH₂)_(n)CH₃]₂ or —CN where n=0 to 5; R₁₅ is H oralkyl of 1 to 10 carbons; R₁₆ is alkyl of 1 to 10 carbons, and thevalence bond designated a symbolizes covalent attachment of thefluorophore to the linker K.
 34. A phosphoramidite reagent for preparingan oligonucleotide-fluorophore-quencher conjugate, the reagent selectedfrom the group consisting of the structures designated PA-4, PA-5 andPA-6,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN; —OR_(nn), —SR_(nn), —OR_(nn, —NHR) _(nn),—N[R_(nn)]₂ whereR_(nn) is independently H, a blocking group compatible with oligomersynthesis removable under acid or alkaline conditions; or an alkyl oralkanoyl group having 1 to 10 carbon atoms; j and k independently are 1to 10, R₁₀ and R₁₁ independently are H

—OR₁₂, —NHR₁₃, halogen,—O(CH₂) _(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃,—C(═O)NH₂, —N[(CH₂)_(n)CH₃]₂, O-alkyl or O-alkanoyl where the

alkanoyl gro

has 1 to 10 carbons, or —CN where n=0 to 5; h=1 to 20; and R₁₂ and R₁₃are blocking groups compatible with ODN synthesis; R₁₅ is H or alkyl of1 to 10 carbons; R₁₆ is alkyl of 1 to 10 carbons.
 35. A phosphoramiditereagent in accordance with claim 34 that has the formula designatedPA-4.
 36. A phosphoramidite reagent in accordance with claim 35 where R₈is —OC(O)CH(CH₃)₂, R₉ is H, j is 2 and k is
 6. 37. A phosphoramiditereagent in accordance with claim 34 that has the formula designatedPA-5.
 38. A phosphoramidite reagent in accordance with claim 37 whereR₁₀ is OC(O)CH(CH₃)₂, R₁₁ is H and h is
 3. 39. A phosphoramidite reagentin accordance with claim 34 that has the formula designated PA-6.
 40. Aphosphoramidite reagent in accordance with claim 39 where R₁₅ is methyland R₁₆ is n-propyl.
 41. A phosphoramidite reagent for preparing anoligonucleotide-fluorophore-quencher conjugate, the reagent having theformula

wherein R₀, R₁, R₂, R₃ and R₄ are independently —H, halogen,—O(CH₂)_(n*)CH₃, —(CH₂)_(n*)CH₃ where n*=0 to 5, —NO₂, —SO₃, —N[(CH₂)_(n′)CH₃]₂where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n″)CH₃ where n″=0 to 5; nis 1 to 10; q is 1 to 20, and X₂ is H or dimethoxytrityl, methoxytrityl,trityl or an acid labile blocking group.
 42. A phosphoramidite reagentin accordance with claim 41 where R_(o) is H, R₁ is NO₂ in the 4position of the benzene nucleus, R₂ is Cl in the 2 position of thebenzene nucleus, and R₃ and R₄ are hydrogen, R₅ is ethyl, n is 1 and qis
 2. 43. An oligonucleotide conjugate having the formula FL-ODN-MGBwhere ODN is an oligonucleotide or nucleic acid; MGB is minor groovebinder moiety covalently attached to the ODN moiety or to the quenchermoiety through a linker having the length of 0 to approximately 30atoms; FL is a fluorophore covalently attached to the ODN through alinker having the length of 0 to approximately 30 atoms, saidfluorophore moiety having the structure selected from the groupdesignated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN;—OR_(nn), —SR_(nn), —OR_(nn),—NHR_(nn,—N[R) _(nn)]₂ where R_(nn)is independently H, an alkyl group of 1 to 10 carbons or an alkanoylgroup of 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons, R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker.
 44. Anoligonucleotide conjugate in accordance with claim 43 where thefluorophore has the formula designated FL-1.
 45. An oligonucleotideconjugate in accordance with claim 44 where R₈ is —OC(O)CH(CH₃)₂ and R₉is H.
 46. An oligonucleotide conjugate in accordance with claim 43 wherethe fluorophore has the formula designated FL-2.
 47. An oligonucleotideconjugate in accordance with claim 46 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁is H.
 48. An oligonucleotide conjugate in accordance with claim 43 wherethe fluorophore has the formula designated FL-3.
 49. An oligonucleotideconjugate in accordance with claim 49where R₁₅ is methyl and R₁₆ isn-propyl.
 50. An oligonucleotide conjugate having the formula FL-ODNwhere ODN is an oligonucleotide or nucleic acid; FL is a fluorophorecovalently attached to the ODN through a linker having the length of 0to approximately 30 atoms, said fluorophore moiety having the structureselected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN; —OR_(nn), —SR_(nn), —OR_(nn), —NHR_(nn),—N[R_(nn)]₂ whereR_(nn), is independently H, an alkyl group of 1 to 10 carbons or an-alkanoyl group of 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN,—OR₁₂, —N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃,—C(=O)NH₂, —N[(CH₂)_(n)CH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10carbons alkanoyl of 1 to 10 carbons,; R₁₅ is H or alkyl of 1 to 10carbons; R₁₆ is alkyl of 1 to 10 carbons, and the valence bonddesignated a symbolizes covalent attachment of the fluorophore to thelinker.
 51. An oligonucleotide conjugate in accordance with claim 50where the fluorophore has the formula designated FL-1.
 52. Anoligonucleotide conjugate in accordance with claim 51 where R₈ isOC(O)CH(CH₃)₂ and R₉ is H.
 53. An oligonucleotide conjugate inaccordance with claim 50 where the fluorophore has the formuladesignated FL-2.
 54. An oligonucleotide conjugate in accordance withclaim 53 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.
 55. An oligonucleotideconjugate in accordance with claim 50 where the fluorophore has theformula designated FL-3.
 56. An oligonucleotide conjugate in accordancewith claim 55 where R₁₅ is methyl and R₁₆ is n-propyl.
 57. A method forhybridizing nucleic acids, comprising the steps of: (a) providing afirst nucleic acid and a second nucleic acid, (b) incubating the nucleicacids under hybridization conditions, and (c) identifying hybridizednucleic acids; wherein at least one of the nucleic acids comprises aFL-nucleic-acid-Q conjugate where FL is a fluorophore moiety covalentlyattached to the nucleic acid through a linker having the length of 0 toapproximately 30 atoms, and Q is a quencher moiety covalently attachedto the nucleic acid through a linker having the length of 0 toapproximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —H,halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n″)CH₃where n″=0 to 5, and where the quencher moiety is attached to the linkerthrough the valence bond designated a.
 58. A method in accordance withclaim 57 where in the formula Q of the quencher moiety R_(o) is H, R₁ isNO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 positionof the benzene nucleus, and R₃ and R4 are hydrogen and R₅ is ethyl. 59.A method for hybridizing nucleic acids, comprising the steps of: (a)providing a first nucleic acid and a second nucleic acid, (b) incubatingthe nucleic acids under hybridization conditions, and (c) identifyinghybridized nucleic acids; wherein at least one of the nucleic acidscomprises a FL-nucleic-acid-Q conjugate where Q is a quencher moietycovalently attached to the nucleic acid through a linker having thelength of 0 to approximately 30 atoms, and wherein FL is a fluorophorecovalently attached to the ODN through a linker having the length of 0to approximately 30 atoms, said fluorophore moiety having the structureselected from the group designated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN;—OR_(nn), —SR_(nn), —OR_(nn), —NHR_(nn),—N[R_(nn)]₂ where R_(nn)is independently H, an alkyl group of 1 to 10 carbons or an alkanoylgroup of 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons,; R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker.
 60. Amethod in accordance with claim 59 where the fluorophore has the formuladesignated FL-1.
 61. A method in accordance with claim 60 where R₈ isOC(O)CH(CH₃)₂ and R₉ is H.
 62. A method in accordance with claim 59where the fluorophore has the formula designated FL-2.
 63. A method inaccordance with claim 62 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.
 64. Amethod in accordance with claim 59 where the fluorophore has the formuladesignated FL-3.
 65. A method in accordance with claim 64 where R₁₅ ismethyl and R₁₆ is n-propyl.
 66. A method for discriminating betweenpolynucleotides which differ by a single nucleotide, the methodcomprising the following steps: (a) providing a polynucleotidecomprising a target sequence, (b) providing at least two FL-ODN-Qconjugates, wherein ODN represents an oligonucleotide moiety, one of theat least two FL-ODN-Q conjugates has a sequence that is perfectlycomplementary to the target sequence and at least one other of theFL-ODN-Q conjugates has a single-nucleotide mismatch with the targetsequence; (c) separately incubating each of the FL-ODN-Q conjugates withthe polynucleotide under hybridization conditions; and (d) determiningthe hybridization strength between each of the FL-ODN-Q and thepolynucleotide, wherein FL is a fluorophore moiety covalently attachedto the nucleic acid through a linker having the length of 0 toapproximately 30 atoms, and Q is a quencher moiety covalently attachedto the nucleic acid through a linker having the length of 0 toapproximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R are independently —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n″)CH₃where n″=0 to 5, and where the quencher moiety is attached to the linkerthrough the valence bond designated a.
 67. A method in accordance withclaim 66 where in the formula of the quencher moiety Q R_(o) is H, R₁ isNO₂ in the 4 position of the benzene nucleus, R₂ is Cl in the 2 positionof the benzene nucleus, and R₃ and R₄ are hydrogen and R₅ is ethyl. 68.A method for discriminating between polynucleotides which differ by asingle nucleotide, the method comprising the following steps: (a)providing a polynucleotide comprising a target sequence, (b) providingat least two FL-ODN-Q conjugates, wherein ODN represents anoligonucleotide moiety, one of the at least two FL-ODN-Q conjugates hasa sequence that is perfectly complementary to the target sequence and atleast one other of the FL-ODN-Q conjugates has a single-nucleotidemismatch with the target sequence; (c) separately incubating each of theFL-ODN-Q conjugates with the polynucleotide under hybridizationconditions; and (d) determining the hybridization strength between eachof the FL-ODN-Q and the polynucleotide, wherein Q is a quencher moietycovalently attached to the nucleic acid through a linker having thelength of 0 to approximately 30 atoms, and FL is a fluorophore moietycovalently attached to the nucleic acid through a linker having thelength of 0 to approximately 30 atoms, and the fluorophore moiety havingthe structure selected from the group designated FL-1, FL-2 and FL-3,

wherein R₈and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂, or—CN;—OR_(nn), —SR_(nn), —OR_(nn), —NHR_(nn),—N[R_(nn)]₂ where R_(nn) isindependently H, an alkyl group of 1 to 10 carbons or an alkanoyl groupof 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons; R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker.
 69. Amethod in accordance with claim 68 where the fluorophore has the formuladesignated FL-1.
 70. A method in accordance with claim 69 where R₈ isOC(O)CH(CH₃)₂ and R₉ is H.
 71. A method in accordance with claim 68where the fluorophore has the formula designated FL-2.
 72. A method inaccordance with claim 71 where R₁₀ is OC(O)CH(CH₃)₂ and R₁₁ is H.
 73. Amethod in accordance with claim 68 where the fluorophore has the formuladesignated FL-3.
 74. A method in accordance with claim 73 where R₁₅ ismethyl and R₁₆ is n-propyl.
 75. A method for hybridizing nucleic acids,comprising the steps of: (a) providing a first nucleic acid and a secondnucleic acid, (b) incubating the nucleic acids under hybridizationconditions, and (c) identifying hybridized nucleic acids; wherein atleast one of the nucleic acids comprises a FL-nucleic-acid-Q-MGBconjugate where FL is a fluorophore moiety covalently attached to thenucleic acid through a linker having the length of 0 to approximately 30atoms, MGB is minor groove binder moiety covalently attached to the ODNmoiety or to the quencher moiety through a linker having the length of 0to approximately 30 atoms and Q is a quencher moiety covalently attachedto the nucleic acid through a linker having the length of 0 toapproximately 30 atoms, the quencher moiety having the structure

where R₀, R₁, R₂, R₃ and R₄ are independently —H,halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃ where n=0 to 5, —NO₂, —SO₃,—N[(CH₂)_(n′)CH₃]₂ where n′=0 to 5 or —CN, and R₅=—H or —(CH₂)_(n″)CH₃where n″=0 to 5, and where the quencher moiety is attached to the linkerthrough the valence bond designated a.
 76. A method for hybridizingnucleic acids, comprising the steps of: (a) providing a first nucleicacid and a second nucleic acid, (b) incubating the nucleic acids underhybridization conditions, and (c) identifying hybridized nucleic acids;wherein at least one of the nucleic acids comprises a FL-ODN-Q-MGBconjugate where ODN is a nucleic acid or modified nucleic acid, MGB isminor groove binder moiety covalently attached to the ODN moiety or tothe quencher moiety through a linker having the length of 0 toapproximately 30 atoms, Q is a quencher moiety covalently attached tothe ODN through a linker having the length of 0 to approximately 30atoms, and FL is a fluorophore moiety covalently attached to the nucleicacid through a linker having the length of 0 to approximately 30 atoms,and the fluorophore moiety having the structure selected from the groupdesignated FL-1, FL-2 and FL-3,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN;—OR_(nn), —SR_(n), —OR_(nn),—NHR_(nn),—N[R_(nn)]₂ where R_(nn) isindependently H, an alkyl group of 1 to 10 carbons or an alkanoyl groupof 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5,or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons, R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker; and Qcomprises a diazo moiety having the formula:

wherein covalent attachment to the linker is through the nitrogen atomdesignated as b.
 77. A method for hybridizing nucleic acids, comprisingthe steps of: (a) providing a first nucleic acid and a second nucleicacid, (b) incubating the nucleic acids under hybridization conditions,and (c) identifying hybridized nucleic acids; wherein at least one ofthe nucleic acids comprises a FL-ODN-Q-MGB conjugate where ODN is anucleic acid or modified nucleic acid, MGB is minor groove binder moietycovalently attached to the ODN moiety or to the quencher moiety througha linker having the length of 0 to approximately 30 atoms, Q is aquencher moiety covalently attached to the ODN through a linker havingthe length of 0 to approximately 30 atoms, and FL is a fluorophoremoiety covalently attached to the nucleic acid through a linker havingthe length of 0 to approximately 30 atoms,, and the fluorophore moietyhaving the structure selected from the group designated FL-1, FL-2 andFL-3,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN;—OR_(nn), —SR_(nn), —OR_(nn),—NHR_(nn),—N[R_(nn)]₂ where R_(nn)is independently H, an alkyl group of 1 to 10 carbons or an alkanoylgroup of 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5 or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons, R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker.
 78. Amethod for hybridizing nucleic acids, comprising the steps of: (a)providing a first nucleic acid and a second nucleic acid, (b) incubatingthe nucleic acids under hybridization conditions, and (c) identifyinghybridized nucleic acids; wherein at least one of the nucleic acidscomprises a FL-ODN-Q conjugate where ODN is a nucleic acid or modifiednucleic acid, Q is a quencher moiety covalently attached to the ODNthrough a linker having the length of 0 to approximately 30 atoms, andFL is a fluorophore moiety covalently attached to the nucleic acidthrough a linker having the length of 0 to approximately 30 atoms, andthe fluorophore moiety having the structure selected from the groupdesignated FL-1, FL-2 and FL-3 ,

wherein R₈ and R₉ independently are H, halogen, —NO₂, —SO₃, —C(═O)NH₂,or —CN;—OR_(nn), —SR_(nn), —OR_(nn), —NHR_(nn),—N[R_(nn) ₂ where R_(nn)is independently H, an alkyl group of 1 to 10 carbons or an alkanoylgroup of 1 to 10 carbons; R₁₀ and R₁₁ independently are H, —CN, —OR₁₂,—N(R₁₂)₂, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, —SO₃, —C(═O)NH₂,—N[(CH₂)_(n)CH₃]₂ where n=0 to 5, or R₁₂ is alkyl of 1 to 10 carbonsalkanoyl of 1 to 10 carbons, R₁₅ is H or alkyl of 1 to 10 carbons; R₁₆is alkyl of 1 to 10 carbons, and the valence bond designated asymbolizes covalent attachment of the fluorophore to the linker; and Qcomprises a diazo moiety having the formula:

wherein covalent attachment to the linker is through the nitrogen atomdesignated as b.